Nejmoa2031054 Protocolcrisprsicklecell

Protocol
This trial protocol has been provided by the authors to give readers additional information about their work.
Protocol for: Frangoul H, Altshuler D, Cappellini MD, et al. CRISPR-Cas9 gene editing for sickle cell disease and
β-thalassemia. N Engl J Med 2021;384:252-60. DOI: 10.1056/NEJMoa2031054
This supplement contains the following:
Clinical Study Protocol CTX001-111

1. Original protocol (Version 1.0) – p2
2. Final protocol (Version 5.0) – p91
3. Summary of changes – p95
Clinical Study Protocol CTX001-121
1. Original protocol (Version 1.0) – p183

2. Final protocol (Version 5.0) – p263
3. Summary of changes – p264
CRISPR Therapeutics AG Clinical Study Protocol
CRISPR-Cas9 Modified CD34+ hHSPCs CTX001-111
PROTOCOL ACCEPTANCE FORM
Protocol: CTX001-111
Title: A Phase 1/2 Study of the Safety and Efficacy of a Single Dose of
Autologous CRISPR-Cas9 Modified CD34+ Human Hematopoietic Stem
and Progenitor Cells (hHSPCs) in Subjects with Transfusion-Dependent
β-Thalassemia
Date: November 22, 2017
Amendment: N/A
I have carefully read this protocol and agree that it contains all of the necessary information
required to conduct this study. I agree to conduct this study as described and according to the
Declaration of Helsinki, International Conference on Harmonisation (ICH) Guidelines for Good
Clinical Practice (GCP), and all applicable regulatory requirements.
Investigator’s Signature Date
Name (printed)
Version 1.0 3 Confidential

SYNOPSIS
Name of Sponsor/Company:
CRISPR Therapeutics AG
Name of Investigational Product:
CTX001
Title of Study:
CTX001-111
A Phase 1/2 Study of the Safety and Efficacy of a Single Dose of Autologous CRISPR-Cas9 Modified CD34+
Human Hematopoietic Stem and Progenitor Cells (hHSPCs) in Subjects with Transfusion-Dependent
β-Thalassemia
Study Centers: Number of Subjects:
Approximately 8 Up to 30
Study Period: Phase of Development:
Screening through Infusion = ~ 3-6 months 1/2
Follow-up = 24 months (2 years)
Study Rationale:
The purpose of the study is to evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
+
modified CD34 Human Hematopoietic Stem and Progenitor Cells (hHSPCs) in subjects ≥18 and ≤35 years of
age with transfusion-dependent β-thalassemia (TDT). CRISPR-Cas9 editing of autologous CD34+ hHSPCs at
erythroid lineage specific enhancer of BCL11A is intended to disrupt BCL11A gene expression in erythroid cells
and consequently increase γ-globin expression in subjects with TDT. The increase in γ-globin is expected to
recreate a state similar to the elevated γ-globin and fetal hemoglobin (HbF) observed in people with Hereditary
Persistence of Fetal Hemoglobin (HPFH) to ameliorate the symptoms and signs of TDT.
Study Objectives:
Primary
+
• To evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9 modified CD34
hHSPCs (CTX001) in subjects with TDT
Secondary
• To quantify percentage of edited alleles in peripheral blood leukocytes and bone marrow cells
• To assess the production of HbF post-CTX001 infusion
• To assess the effects of infusion of CTX001 on disease-specific events and clinical status
Exploratory
• To assess the ability of biomarkers to characterize CTX001 effect and predict treatment outcomes
Study design:
This is a single-arm, open-label, multisite, single-dose Phase 1/2 study that will enroll subjects 18 to 35 years of
age with non-β0/β0 TDT. Transfusion dependence is defined as history of at least 100 mL/kg/year or 10 units/year
of packed red blood cell (RBC) transfusions in the 2 years prior to signing of informed consent. The first part of
the study will include up to 12 subjects. The study may be expanded to include an additional 18 subjects
(expansion cohort).
The study will be conducted in 4 stages:
Stage 1: Screening and pre-mobilization period
During this period, subjects who meet the inclusion/exclusion criteria have the option to undergo fertility
preservation via cryopreservation of oocyte or sperm.
+
Stage 2: Mobilization, autologous CD34 stem cell collection, CTX001 manufacture and disposition
After eligibility has been confirmed, each subject will undergo stem cell mobilization with a combination of
granulocyte-colony stimulating factor (G-CSF; e.g. filgrastim) and plerixafor. Peripheral blood mononuclear cells

(PBMCs) will be collected by apheresis. Subjects will undergo apheresis for 2 or 3 consecutive days to collect
+ + +
CD34 HSPC. The targeted CD34 cell collection is at least 15 x 106 CD34 cells/kg for manufacturing of
6 +
CTX001. An additional 2 x 10 CD34 cells/kg will be collected as backup for rescue therapy in an event of non-
engraftment with CTX001. All collected cells intended for manufacturing will be shipped daily at 2-8oC to the
manufacturing facility. Cells collected for rescue therapy will not undergo the editing manufacturing process and
will be cryopreserved at the site.
If sufficient numbers of cells for CTX001 manufacturing and backup are not obtained (as determined based on
discussion between the Sponsor and investigator), a second mobilization and apheresis cycle may be performed at
least 14 days after the first day of initial mobilization cycle.
Stage 3: Myeloablative conditioning (Stage 3A) and infusion of CTX001 (Day 1, Stage 3B)
Stage 3A - Myeloablative conditioning: After the CTX001 product is received at the site and it has been
confirmed that the backup cells have been stored; the subject will be hospitalized to undergo myeloablative
conditioning with busulfan.
Busulfan will be administered intravenously (IV) daily at a starting dose of 3.2 mg/kg/day for 4 consecutive days.
Once-daily dosing is the preferred schedule; however, the busulfan dosing regimen may be adjusted to be given
every 6 hours (Q6H) per a site’s standard practice.
The dose of busulfan will be adjusted based on the pharmacokinetics (PK) of the first busulfan dose to maintain
appropriate levels for myeloablation. The average target area under the curve (AUC) for subjects receiving a
starting dose of 3.2 mg/kg/day for 4 days is 5000 µM*min (range: 4500 to 5500); equivalent to target cumulative
busulfan exposure of 90 mg x hr/L (range 80-100 mg x hr/L). The mean AUC for subjects administered busulfan
Q6H for 4 days is 1125 µM*min (range: 900 to 1350).
Clinical sites may use a test dose of busulfan several days before beginning myeloablation to pre-determine
busulfan dose. Prophylaxis of veno-occlusive disease using defibrotide can be used per investigator’s discretion
in accordance with standard of care.
Stage 3B - CTX001 infusion:
Infusion of CTX001 should occur at a minimum of 48 hours following the completion of busulfan infusion and at
a maximum of 7 days after the completion of busulfan infusion.
+
On Day 1, after thawing, the entire dose of CTX001, which at a minimum will be 3.0 × 106 CD34 cells/kg, will
be administered through a central venous catheter. All vial(s) containing CTX001 should be infused.
Stage 4: Follow-up, through engraftment and up to 24 months after CTX001 product infusion
Stage 4A - Post-infusion until hospital discharge engraftment): Subjects will be followed daily in the transplant
unit and receive supportive care according to standard practices for subjects undergoing hematopoietic stem cell
transplant (HSCT). Subjects will be monitored for adverse events (AEs) and engraftment. Packed RBC and
platelet transfusions will be given to subjects per standard practices for patients undergoing HSCT. Subjects will
be discharged from the transplant unit upon neutrophil engraftment (defined as absolute neutrophil count [ANC]
≥500/µL for 3 consecutive days) and stabilization of major medical issues as per local hospital guidelines.
Stage 4B - Post-engraftment to 2-years post-infusion: Subjects will be followed for 24 months after CTX001
infusion with physical exams, laboratory and imaging assessments, and AE evaluations. Subjects will be allowed
to restart iron chelation approximately 1 month after CTX001 infusion according to the site’s management
guidelines and based on the severity of the subject’s pre-treatment iron overload.
Following engraftment, transfusions of packed RBCs should be avoided for hemoglobin (Hb) ≥9 g/dL, unless
medically indicated (e.g., symptomatic anemia or as a requirement for surgery). It is recommended that subjects
should receive packed RBC transfusions for Hb <7.0 g/dL, while medical judgement is advised to transfuse for
Hb levels of 7-9 g/dL based on a subject’s clinical needs.
Data Monitoring Committee: A Data Monitoring Committee (DMC) comprised of members with appropriate
scientific and medical expertise to monitor the study will be convened. The DMC will be charged with ensuring
the safety of the subjects. The DMC may recommend that the Sponsor suspend enrollment, amend the study, or
discontinue the study at any time.
Number of Subjects and Enrollment Plan: In the first part of the study, 12 adult non-β0/β0 TDT subjects will
be enrolled and treated with the CTX001. Replacement subjects may be added if subjects fail screening,

withdraw consent prior to CTX001 infusion, or do not receive CTX001 infusion for any other reason.
The first two subjects will be treated with CTX001 in a staggered manner. The second subject will not undergo
myeloablation until the first subject achieves neutrophil engraftment (ANC ≥500/µL x 3 consecutive days) and
the available engraftment and safety data have been reviewed by the DMC. Once the second subject has achieved
neutrophil engraftment and has not experienced Grade≥3 AEs other than those typically associated with busulfan
conditioning or autologous transplant procedure, the remaining subjects can undergo conditioning and CTX001
infusion concurrently. In the event that the second subject infused with CTX001 experiences Grade 3≥AEs other
than those typically associated with busulfan conditioning or autologous transplant procedure, a DMC meeting
will be convened and data will be reviewed before the remaining subjects can undergo conditioning and CTX001
infusion. All steps that precede busulfan myeloablation such as consent, screening, and stem cell collection may
proceed concurrently without staggering subjects.
After or more subjects have been treated with CTX001, the trial may be expanded to an additional 18 subjects
(expansion cohort) upon review of available safety and efficacy data and agreement from the DMC.
All subjects infused with CTX001 will be asked to enroll into an additional long-term follow-up study or registry.
Inclusion Criteria:
1. Age ≥18 and ≤35 years of age.
2. Able to provide written informed consent.
3. Diagnosis of transfusion-dependent β-thalassemia (TDT) as defined by:
a. Documented homozygous β-thalassemia (with the exception of the β0/β0 genotype) or compound
heterozygous β-thalassemia including β-thalassemia/hemoglobin E (HbE). Subjects can be enrolled
based on historical data, but a confirmation of the genotype using the study central laboratory will be
required before busulfan conditioning. The β0/β0 genotypes are defined using the HbVar Database.
b. A history of at least 100 mL/kg/year or 10 units/year of packed RBC transfusions in the prior 2 years
before signing the consent.
4. Karnofsky performance status of ≥80%.
5. Eligible for autologous stem cell transplant as per investigator’s judgment.
6. Access to detailed medical records on packed RBC transfusions, including volume or units of packed RBCs
and associated pre-transfusion Hb values, and in-patient hospitalizations, for at least the 2 years prior to
consent.
7. Female subjects of childbearing potential (postmenarcheal, has an intact uterus and at least 1 ovary, and is
less than 1 year postmenopausal) must agree to use acceptable method(s) of contraception from consent
through at least 6 months after CTX001 infusion.
8. Male subjects must agree to use effective contraception (including condoms) from start of busulfan
conditioning through at least 6 months after CTX001 infusion.
9. Willing to participate in an additional long-term follow-up study or registry after completion of this study.
Exclusion Criteria:
1. An available 10/10 human leukocyte antigen (HLA)-matched related donor.
2. Prior allogeneic HSCT.
3. Subjects with associated α-thalassemia and >1 alpha chain deletion.
4. Subjects with a β0/β0 thalassemia genotype or sickle cell beta thalassemia variant.
5. Clinically significant and active bacterial, viral, fungal, or parasitic infection as determined by the
investigator.
6. White blood cell count <3 × 109/L or platelet count <50 × 109/L not related to hypersplenism.
7. History of a significant bleeding disorder.
8. History of any illness or any clinical condition that, in the opinion of the investigator, might confound the
results of the study or pose an additional risk in administering study drug to the subject. This may include,
but is not limited to: history of relevant drug allergies; history of cardiovascular including heart failure or
central nervous system disease; history or presence of clinically significant pathology; or history of mental

disease.
9. Any prior or current malignancy or myeloproliferative disorder or a significant immunodeficiency disorder.
10. Advanced liver disease, defined as:
a. Aspartate transaminase, alanine transaminase >3 x the upper limit of normal (ULN), or direct bilirubin
value >2 x the ULN, or:
b. Baseline prothrombin time (International Normalized Ratio; INR) >1.5 x ULN, or
c. History of cirrhosis or any evidence of bridging fibrosis, or active hepatitis on liver biopsy. Liver Biopsy
is required when liver iron concentration (LIC) is ≥15 mg/g on T2* magnetic resonance imaging (MRI)
of liver. If a liver biopsy has been performed less than 6 months prior to consent, it does not need to be
repeated.
11. A cardiac T2* <10 ms by MRI or left ventricular ejection fraction (LVEF) <45% by echocardiogram.
12. Baseline estimated glomerular filtration rate <60 mL/min/1.73 m2.
13. Diffusion capacity of the lungs for carbon monoxide (DLco) <50% of predicted (corrected for hemoglobin
and/or alveolar volume).
14. Prior treatment with gene therapy.
15. Intolerance or known sensitivity to plerixafor, G-CSF products (e.g., filgrastim), or busulfan. Prior
anaphylaxis with excipients of CTX001 product (dimethyl sulfoxide [DMSO], Dextran).
16. Positive serology for human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus-2 (HIV-
2), hepatitis B virus (HBV) (Hepatitis B core antibody [HBcAb] and positive HBV PCR), or hepatitis C virus
(HCV) (positive HCV PCR). Positive serology for syphilis, toxoplasmosis or any other infectious disease
marker as required by local testing for cellular processing.
17. Participation in another clinical study with an investigational drug within 30 days of Screening or fewer than
5 half-lives of the investigational agent, whichever is longer from Screening.
18. An assessment by the investigator that the subject would not comply with the study procedures outlined in
the protocol.
19. Pregnant or breastfeeding females.
Duration of Subject Participation: Duration of Stage 1 will be approximately 1-3 months; Stage 2
approximately 2-3 months; Stage 3 approximately 1 month; Stage 4 approximately 2 years. Subjects will be
followed on study for a total of approximately 2.5 years after consent.
After completing the study, all subjects will be asked to participate in a long-term follow-up study or registry.
+
Test Product, Dose, and Mode of Administration: CTX001 product is composed of autologous CD34
hHSPCs modified with CRISPR-Cas9 at the BCL11A gene locus in order to increase gamma-globin/HbF.
CTX001 is suspended in a cryopreservative solution containing dimethyl sulfoxide (DMSO) and Dextran-40 in
the final container for the intended medical use. Subjects will receive the entire dose of CTX001, which will be at
+
a minimum of 3.0 × 106 CD34 cells/kg, through a central venous catheter.
Study Endpoints
Safety:
• Proportion of subjects with engraftment. Engraftment is defined as ANC ≥500/µL for three consecutive days.
Engraftment failure is defined as any subject not achieving neutrophil engraftment by Day +42 following
+
CTX001 infusion or if backup unmodified CD34 cells are utilized.
• Time to engraftment.
• Frequency and severity of collected AEs from signing of informed consent through Month 24 visit as
assessed by the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events
(CTCAE) v4.03.
• Incidence of transplant related mortality (TRM) at 100 days and 1 year post-CTX001 infusion. TRM is
defined as death possibly related to the transplantation procedure as assessed by the investigator.
• All-cause mortality.

Primary Efficacy
• Proportion of subjects achieving transfusion reduction for at least 6 months (TR6).
Key Secondary Efficacy
• Proportion of subjects achieving transfusion independence for at least 6 months (TI6).
Secondary Endpoints
• Proportion of alleles with intended genetic modification present in peripheral blood leukocytes.
• Proportion of alleles with intended genetic modification present in bone marrow cells.
• Change in fetal hemoglobin concentration (pre-transfusion) over time.
• Change in health-related quality of life (HRQoL) from baseline over time using EQ-5D-5L and FACT-BMT.
• Change in parameters of iron overload, including:
o Liver iron concentration (LIC) and cardiac iron content (CIC) from baseline as assessed by T2*
MRI.
o Change in serum ferritin level from baseline over time.
• Proportion of subjects receiving iron chelation therapy over time.
Exploratory Endpoints
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) from baseline (pre-
transfusion) over time.
• Expression of α-globin and non-α-globin mRNA in circulating reticulocytes over time.
• Change in erythropoietin (EPO) concentrations over time.
• Change in hepcidin concentrations over time.
• Assessment of erythropoiesis on bone marrow analysis compared with baseline over time.
Statistical Methods:
Statistical analysis details will be provided in the Statistical Analysis Plan (SAP), which will be finalized before
the clinical data lock for the study.
For the primary efficacy and key secondary endpoints, TR6 and TI6 will be estimated as proportions with the
corresponding 2-sided 95% exact Clopper-Pearson confidence. For example, if 20 of 30 subjects treated with
CTX001 achieve TR6, the response rate of TR6 will be estimated as 66.7% with the exact 95% confidence
interval of (47.2%, 82.7%).
Analysis Populations
• The enrolled set includes all subjects who sign the informed consent and meet the inclusion/exclusion criteria
• The full analysis set (FAS) is a subset of the enrolled set that includes subjects who receive CTX001
infusion. The efficacy analyses will be performed based on the FAS. Demographics and baseline
characteristics will be summarized based on FAS as well.
• The safety analysis set is a subset of the enrolled set that includes subjects who start the mobilization
regimen. Analyses of the safety assessments will be based on the safety analysis set. Collected AEs
(including but not limited to all study procedure-related AEs and all SAEs from the signing of the ICF to
start of busulfan conditioning; and all AEs and all SAEs from start of busulfan conditioning through month
24 after CTX001 infusion, engraftment, etc.), labs, vital signs, ECG assessments will be summarized.

TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES

TABLE OF CONTENTS
TITLE PAGE .................................................................................................................................1
PROTOCOL APPROVAL SIGNATURE PAGE ......................................................................2
PROTOCOL ACCEPTANCE FORM .........................................................................................3
SYNOPSIS ......................................................................................................................................4
TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES ..............................9
LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS ..........................................14
1. INTRODUCTION ..............................................................................................................18
1.1. β-thalassemia ...................................................................................................................18
1.2. Current Treatment Approaches for Transfusion-Dependent β-Thalassemia
(TDT) .......................................................................................................................20
1.3. CRISPR-Cas9 Gene Editing Approach ...........................................................................21
1.4. BCL11A ..........................................................................................................................23
1.5. Investigational Product: CTX001 ....................................................................................23
1.6. Non-clinical Experience ..................................................................................................24
1.7. Clinical Experience .........................................................................................................25
1.8. Therapeutic Rationale ......................................................................................................25
1.9. Dose Rationale.................................................................................................................26
1.10. Risk Assessment .........................................................................................................27
1.10.1. Potential Risks of CTX001 ..................................................................................27
1.10.2. Potential Risks of Mobilization, Apheresis and Autologous
Transplantation ..........................................................................................28
2. STUDY OBJECTIVES AND ENDPOINTS ....................................................................29
2.1. Objectives ........................................................................................................................29
2.1.1. Primary ................................................................................................................29
2.1.2. Secondary ............................................................................................................29
2.1.3. Exploratory ..........................................................................................................29
2.2. Endpoints .........................................................................................................................29
2.2.1. Safety Endpoints ..................................................................................................29
2.2.2. Primary Efficacy Endpoint ..................................................................................30
2.2.3. Key Secondary Efficacy Endpoint ......................................................................30
2.2.4. Secondary Endpoints ...........................................................................................30
2.2.5. Exploratory Endpoints .........................................................................................30
3. STUDY POPULATION .....................................................................................................31
3.1. Number of Subjects .........................................................................................................31
3.2. Subject Inclusion Criteria ................................................................................................31
3.3. Subject Exclusion Criteria ...............................................................................................32
3.4. Enrolled Subjects and Screen Failures ............................................................................33

3.5. Waivers of Inclusion/Exclusion Criteria .........................................................................33

3.6. Contraception ..................................................................................................................33
3.7. Subject Identification.......................................................................................................35
3.8. Subject Withdrawal .........................................................................................................36
3.9. Replacement of Subjects .................................................................................................36
4. STUDY DESIGN ................................................................................................................36
4.1. Study Overview ...............................................................................................................36
4.2. Study Duration.................................................................................................................39
4.3. Study Enrollment Plan .....................................................................................................39
5. STUDY STOPPING RULES.............................................................................................39
5.1. Enrollment Suspension Criteria .......................................................................................39
5.2. Stopping Rules.................................................................................................................40
5.3. End of Study Definition...................................................................................................41
5.4. Sponsor’s Termination of Study ......................................................................................41
6. TREATMENT PLAN ........................................................................................................41
6.1. Description of CTX001 ...................................................................................................41
6.2. Mobilization and Apheresis .............................................................................................41
6.2.1. Mobilization ........................................................................................................41
6.2.2. Apheresis Procedure ............................................................................................42
6.3. Shipment to Manufacturing Facility................................................................................43
6.4. Manufacture of CTX001 .................................................................................................43
6.5. Shipment of CTX001 Product to Treatment Site ............................................................43
6.6. Storage of CTX001 Product ............................................................................................43
6.7. Conditioning: Busulfan Administration ..........................................................................44
6.8. CTX001 Infusion Procedures, Dose, and Administration ...............................................44
6.9. Post-CTX001 Infusion Infection Prophylaxis and Surveillance .....................................45
6.10. Prior and Concomitant Treatments and Procedures ...................................................45
6.10.1. Prior medications .................................................................................................45
6.10.2. Venous Access.....................................................................................................46
6.10.3. Transfusions ........................................................................................................46
6.10.4. Iron Chelation ......................................................................................................46
6.10.5. Prohibited medications ........................................................................................47
6.11. CTX001 Accountability..............................................................................................47
6.12. Treatment Compliance................................................................................................47
7. VISIT EVALUATION SCHEDULE ................................................................................47
8. STUDY ASSESSMENTS ...................................................................................................54
8.1. Demographic/Medical History ........................................................................................54
8.2. Transfusions ....................................................................................................................54
8.3. Vital Signs and Weight/Height ........................................................................................54
8.4. Physical Examination ......................................................................................................54
8.5. Electrocardiogram ...........................................................................................................54
8.6. Diffusing Capacity of the Lungs for Carbon Monoxide (DLCO) ...................................55
8.7. Imaging Studies ...............................................................................................................55

8.8. Laboratory Assessments ..................................................................................................55

8.9. Blood for Biomarker Assessments ..................................................................................57
8.10. Blood for Exploratory Research Studies and Assay Development ............................57
8.11. Sperm and Oocyte Banking ........................................................................................57
8.12. Biopsies.......................................................................................................................57
8.12.1. Liver Biopsy ........................................................................................................57
8.12.2. Bone Marrow Aspirate ........................................................................................58
8.13. Health-Related Quality of Life (HRQoL)...................................................................58
8.13.1. EQ-5D-5L ............................................................................................................58
8.13.2. FACT-BMT .........................................................................................................58
9. SAFETY MONITORING AND REPORTING ...............................................................59
9.1. Adverse and Serious Adverse Events ..............................................................................59
9.1.1. Definition of Adverse Events ..............................................................................59
9.1.1.1. Adverse Event (AE) ...................................................................................59
9.1.1.2. Clinically Significant Assessments ............................................................59
9.1.1.3. Collection of Adverse Events (AEs) ..........................................................59
9.1.1.4. Serious Adverse Events (SAEs).................................................................62
9.1.1.5. Collection of Serious Adverse Events .......................................................63
9.1.2. Reporting Serious Adverse Events ......................................................................63
9.1.3. Expedited Reporting and Investigator Safety Letters ..........................................64
9.2. Pregnancy ........................................................................................................................64
9.3. Data Monitoring Committee............................................................................................64
10. STATISTICAL AND ANALYTICAL PLANS ...............................................................65
10.1. Sample Size Estimation ..............................................................................................65
10.2. Analysis Set ................................................................................................................65
10.3. General Considerations ...............................................................................................65
10.4. Subject Disposition .....................................................................................................66
10.5. Demographic and Baseline Characteristics ................................................................66
10.6. Efficacy Analysis ........................................................................................................66
10.6.1. Analysis of Primary Efficacy Endpoint ...............................................................66
10.6.2. Analysis of Key Secondary Endpoint..................................................................67
10.6.3. Secondary Efficacy Endpoints ............................................................................67
10.7. Safety Analysis ...........................................................................................................68
10.7.1. Adverse Events ....................................................................................................69
10.7.2. Clinical Laboratory Assessments ........................................................................69
10.7.3. Electrocardiogram ...............................................................................................69
10.7.4. Vital Signs ...........................................................................................................69
10.7.5. Physical Examination ..........................................................................................70
10.8. Exploratory Analysis ..................................................................................................70
10.9. Procedures for Handling Missing Data.......................................................................70
10.10. Steering Committee ..................................................................................................70
10.11. Endpoint Adjudication Committee (EAC) ...............................................................70
11. DATA COLLECTION AND MANAGEMENT..............................................................71
11.1. Monitoring ..................................................................................................................71

11.2. Electronic Data Capture (EDC) ..................................................................................71

11.3. Audits and Inspections ................................................................................................72
12. QUALITY CONTROL AND QUALITY ASSURANCE ...............................................72
13. ETHICAL AND REGULATORY CONSIDERATIONS ...............................................72
13.1. Ethics Committee (EC) / Institutional Review Board (IRB) ......................................72
13.2. Ethics Review .............................................................................................................72
13.3. Conduct of the Study ..................................................................................................73
13.4. Written Informed Consent ..........................................................................................73
14. DATA HANDLING AND RECORDKEEPING .............................................................73
14.1. Inspection of Records .................................................................................................73
14.2. Retention of Records ..................................................................................................73
14.3. Subject Privacy and Confidentiality ...........................................................................74
15. PUBLICATIONS AND CLINICAL STUDY REPORT.................................................75
15.1. Publication of Study Results .......................................................................................75
15.2. Clinical Study Report .................................................................................................75
16. REFERENCES ...................................................................................................................76
17. APPENDICES ....................................................................................................................79
APPENDIX 1. BUSULFAN PK COLLECTION ...........................................................80
APPENDIX 2. KARNOFSKY PERFORMANCE STATUS SCALE ..........................81
APPENDIX 3. EQ-5D-5L QUESTIONNAIRE...............................................................82
APPENDIX 4. FACT-BMT QUESTIONNAIRE ...........................................................86
LIST OF TABLES
Table 1: Abbreviations and Specialist Terms ...........................................................................14
Table 2: Mobilization and Apheresis Timing ...........................................................................42
Table 3: Schedule of Events: Screening (Stage 1) ....................................................................48
Table 4: Schedule of Events: Autologous CD34+ Stem Cell Apheresis (Stage 2) ...................49
Table 5: Schedule of Events: Myeloablative Conditioning and Infusion of CTX001
(Stages 3A/3B)............................................................................................................51
Table 6: Schedule of Events: Post-Infusion through Follow-Up (Stage 4) ..............................52
Table 7: Summary of Clinical Laboratory Tests.......................................................................56
Table 8: Adverse Event (AE) and Serious Adverse Event (SAE) Recording ..........................60
Table 9: Classifications for AE Causality.................................................................................61
Table 10: Grading Scale for Adverse Events ..............................................................................61
Table 11: Classifications for Outcome of an AE ........................................................................62

LIST OF FIGURES
Figure 1 Molecular Structure of Hemoglobin A (OpenStax College, 2016) ............................18
Figure 2 Association between Fetal Hemoglobin Level and Morbidity Score
(Musallam et al., 2012) ...............................................................................................19
Figure 3 Schematic of the CRISPR-Cas9 Complex ...................................................................21
Figure 4 CRISPR-Cas9-Mediated Genome Editing Strategies .................................................22
Figure 5 CTX001 Therapeutic Approach .................................................................................24
Figure 6 Study Schema .............................................................................................................37

LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

The following abbreviations and specialist terms are used in this study protocol.
Table 1: Abbreviations and Specialist Terms
Abbreviation or Specialist Term Explanation

ADL Activities of daily living
AE Adverse event
Allo-HSCT Allogeneic hematopoietic stem cell transplant
ALT Alanine transaminase
ANC Absolute neutrophil count
aPTT Activated partial thromboplastin time
AST Aspartate transaminase
AUC Area under the curve
BM Bone Marrow
bpm Beats per minute
CBC Complete blood count
cGMP Current Good Manufacturing Practice
CIC Cardiac iron content
CRF Case report form
CRISPR-Cas9 CRISPR-Cas9 means: Clustered Regularly Interspaced Short
Palindromic Repeats-CRISPR associated 9 nuclease
crRNA crisprRNA
CTCAE Common Terminology Criteria for Adverse Events
CTX001 Autologous CRISPR-Cas9 modified CD34+ hHSPCs
DLco Diffusing capacity of the lungs for carbon monoxide
DMC Data Monitoring Committee
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DSB Double-strand break
EAC Endpoint Adjudication Committee
EC Ethics Committee
ECG Electrocardiogram
eCRF Electronic case report form


EMA European Medicines Agency
EPO Erythropoietin
EQ-5D-5L EuroQol Questionnaire – 5 dimensions – 5 levels of severity
FACT-BMT Functional assessment of cancer therapy-bone marrow
transplant
FAS Full Analysis Set
FSH Follicle-stimulating hormone
FTA-ABS Fluorescent treponemal antibody absorption test
G-CSF Granulocyte-colony stimulating factor
GCP Good Clinical Practice
GGTP Gamma-glutamyl transpeptidase
GMP Good Manufacturing Practice
GPS (Vertex) Global Patient Safety
Genome-wide Unbiased Identification of DSB Enabled by
GUIDE-seq
sequencing
GvHD Graft-versus-host disease
Hb Hemoglobin
HbA Hemoglobin A or adult hemoglobin
HBB Human B globin
HbcAb Hepatitis B core antibody
HbE Hemoglobin E
HbF Fetal hemoglobin
HBV Hepatitis B virus
HCV Hepatitis C virus
HDR Homology-directed repair
hHSPCs Human hematopoietic stem and progenitor cells
HIV-1 Human immunodeficiency virus-1
HLA Human leukocyte antigen
HPFH Hereditary Persistence of Fetal Hemoglobin
HRQoL Health-related quality of life
HSCT Hematopoietic stem cell transplant
ICF Informed Consent Form


ICH International Conference on Harmonisation
INR International normalized ratio
IRB Institutional Review Board
IV Intravenous / intravenously
kg Kilogram
LDH Lactate dehydrogenase
LIC Liver iron concentration
LT-HSC Long-term hematopoietic stem cells
LVEF Left ventricular ejection fraction
μg Microgram
M Month
Max Maximum
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
MCV Mean corpuscular volume
MedDRA Medical Dictionary for Regulatory Activities
Min Minimum
mL Milliliter
MRI Magnetic resonance imaging
mRNA Messenger ribonucleic acid
N Number of subjects
NCI National Cancer Institute
NHEJ Non-Homologous End-Joining
NSG NOD scid gamma
nt Nucleotide
NTDT Non-transfusion-dependent thalassemia
PAM Protospacer adjacent motif
PBMC Peripheral blood mononuclear cell
PCR Polymerase chain reaction
PFT Pulmonary function test
pH Hydrogen ion concentration
PI Principal Investigator


PK Pharmacokinetic(s)
PT Prothrombin time
PTT Partial thromboplastin time
Q6H Every 6 hours
RBC Red blood cell
RDW Red blood cell distribution width
RNA Ribonucleic acid
RPR Rapid plasma reagin
SAE Serious adverse event
SAP Statistical Analysis Plan
SC Steering Committee
SD Standard Deviation
sgRNA Single-guide RNA
SOP Standard Operating Procedure
SUSAR Suspected unexpected serious adverse reaction
TDT Transfusion-dependent β-thalassemia
TE Treatment emergent
TI Transfusion independence
TIBC Total iron binding capacity
TR Transfusion reduction
TRM Transplant-related mortality
ULN Upper limit of normal
VAS Visual analog scale
VOD Veno-occlusive disease
WBC White blood cell

1. INTRODUCTION
CTX001 is a product consisting of autologous CD34+ hematopoietic stem and progenitor cells
(HSPCs) modified by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR
associated 9 nuclease (CRISPR-Cas9)-mediated gene editing at the erythroid lineage-specific
enhancer region of the B-cell Lymphoma/Leukemia 11A (BCL11A) gene located on intron 2,
between exons 2 and 3, on chromosome 2. The disruption of the BCL11A regulatory sequences
allows for reactivation of transcription and protein expression of γ-globin, resulting in increased
levels of fetal hemoglobin (HbF). In severe β-thalassemia, increased HbF decreases the total
hemoglobin (Hb) deficit in red blood cells (RBCs) and increased γ-globin production improves
the α-globin to non-α-globin ratio imbalance, thereby reducing ineffective erythropoiesis (due to
reduced uncomplexed α-globin chains), and prolonging erythrocyte survival (due to decrease in
hemolysis). This results in improvement of anemia and reduction in transfusion needs in
β-thalassemia patients, similar to a naturally occurring state of Hereditary Persistence of Fetal
Hemoglobin (HPFH).
1.1. β-thalassemia
β-thalassemia is one of the most common autosomal recessive disorders worldwide with high
prevalence in populations in the Mediterranean (5-15%), Middle-East and West Asia (2-5%),
South-East Asia (up to 10%), and South Asia (up to 18%) ( Colah et al., 2010). Due to
population migration, β-thalassemia is also found in Northern Europe, North and South America,
Caribbean, and Australia. Currently, the worldwide living population of β-thalassemia major
patients is estimated to be 200,000 that are registered and receiving treatment (Galanello and
Origa, 2010).
β-thalassemia is caused by a spectrum of mutations that result in reduced or absent production of
adult hemoglobin (HbA). Different forms of Hb are produced during different stages of
development. Fetal hemoglobin (HbF) is the predominant Hb prior to birth and extending into
the newborn period. HbF is a tetrameric globin protein containing 2 γ-globin and 2 α-globin
chains (α2γ2). After the newborn period, the main form of Hb is HbA (Figure 1), a
heterotetramer comprised of 2 β-globin and 2 α-globin chains (α2β2). HbA normally accounts
for > 95% of the total Hb in the blood of adults.
Figure 1 Molecular Structure of Hemoglobin A (OpenStax College, 2016)

improvements in the ineffective erythropoiesis seen in the disease, decreased hemolysis, and
increased total hemoglobin levels from the improved survival of erythrocytes containing higher
levels of HbF. Observational studies have documented that there appears to be no minimum
threshold of HbF that is associated with lower morbidity in patients with β-thalassemia, as any
amount of HbF appeared to be beneficial in one study of non-transfusion-dependent patients with
β-thalassemia intermedia (Musallam et al., 2013). Resultant decrease in ineffective
erythropoiesis due to increased HbF levels may also have a positive effect on iron overload and
end-organ damage (Tanno and Miller, 2010).
1.2. Current Treatment Approaches for Transfusion-Dependent

β-Thalassemia (TDT)
Treatment of TDT includes lifelong blood transfusions every 3-6 weeks. The aim of transfusion
therapy is to keep Hb levels ≥9 g/dL in order to ameliorate the symptoms and physiologic
sequalae of severe anemia and to maintain normal growth and development (Rachmilewitz and
Giardina et al, 2011). Though chronic blood transfusion regimens are effective at preventing the
hallmark symptoms and physical manifestations of disease, they introduce a large iron overload
(Cao et al., 1996) that may lead to mortality through iron associated heart and liver toxicity
(Vichinsky et al., 2005) To prevent this, iron overload must be managed with iron chelation
regimens that are usually initiated at an early age (Saliba et al., 2015). Poor compliance with
chelation regimens remains a key challenge. Despite the improvements with current therapies,
there is poor quality of life and overall survival until the age of 30 years is only 55% (Modell et
al., 2000; Delea et al., 2007).
Currently, the only curative treatment option for TDT is allogeneic hematopoietic stem cell
transplant (allo-HSCT). There are significant risks associated with allo-HSCT such as serious
infections, graft failure and graft-versus-host disease (GvHD), some of which can be fatal. As
such, transplants are infrequently performed, and are offered primarily to subjects who have
available human leukocyte antigen (HLA)-matched sibling donors, who are young (<16 years of
age), and who do not have significant iron overload. Because of the need of a HLA-matched
sibling donor, allo-HSCT is available to only <25% of eligible patients with remainder of the
patients requiring lifelong transfusions and chelation. Transplants using alternative donor sources
such as unrelated cord blood and haploidentical donors remain experimental due to high risk of
engraftment failure and GvHD (Mathews et al., 2014).
The absence of suitable donors, the significant risks associated with transplantation, and the
requirement for post-transplant immunosuppression therapy to prevent GvHD indicate an unmet
medical need for novel therapies with transformative potential for subjects with TDT.
Gene-based therapies are promising approaches that have the potential to provide a functional
cure by increasing HbF levels in subjects with severe β-thalassemia. Additionally, through
genetic modification of autologous hematopoietic stem cells (HSCs), the risk of GvHD and
severe opportunistic infections due to immune suppression associated with allo-HSCT is
avoided.

1.3. CRISPR-Cas9 Gene Editing Approach

The CRISPR-Cas9 system is a naturally occurring defense mechanism in prokaryotes that has
been repurposed as a ribonucleic acid (RNA)-guided deoxyribonucleic acid (DNA)-targeting
platform used for gene editing (Jinek et al., 2012).
The CRISPR-Cas9 system from Streptococcus pyogenes relies on only one protein, the nuclease
Cas9, and two noncoding RNAs, crisprRNA (crRNA) and tracrRNA, to target DNA.
• crRNA drives target sequence recognition and specificity of the CRISPR-Cas9 complex
through Watson-Crick base pairing with a (typically) 20 nucleotide (nt) sequence in the
target DNA. The 5’ 20nt of crRNA is responsible for this DNA recognition. Changing
these 20nt in the crRNA alters the sequence specificity of the CRISPR-Cas9 complex and
allows sequence-specific targeting of other loci (targets).
• TracrRNA hybridizes with the 3’ end of crRNA to form an RNA duplex structure that is
then bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9
complex which can then cleave the target DNA.
These two noncoding RNAs can further be joined by a 4nt linker loop to form a chimeric RNA
called single-guide RNA (sgRNA) (Figure 3). The CRISPR-Cas9 (sgRNA/Cas9) complex
together forms a ribonucleoprotein (RNP).
The CRISPR-Cas9 (sgRNA/Cas9) complex binds double-stranded DNA sequences that contain a
sequence match to the first 20nt of the sgRNA if the target sequence is followed by a protospacer
adjacent motif (PAM).
PAM is a short DNA motif immediately adjacent to the target DNA sequence that the Cas9
endonuclease requires for recognition of the sequence as a target site (Jinek et al., 2012).
Figure 3 Schematic of the CRISPR-Cas9 Complex

Complex containing a single gRNA wherein the crRNA and tracrRNA are joined by a linker loop (adapted from Jinek
et al., 2012).
Once CRISPR-Cas9 (sgRNA/Cas9) complex is bound to DNA at a target site, two independent
nuclease domains in Cas9 will each cleave one of the DNA strands 3 bases upstream of the PAM

site, leaving a blunt end DNA double-strand break (DSB) (Jinek et al., 2012).
Formation of the site-specific DSB is the first of two key steps in genome editing. The second
key step is repair of the DSB. Cells use 2 main DNA repair pathways to resolve the DSB: non-
homologous end-joining (NHEJ) and homology-directed repair (HDR) (Figure 4).
NHEJ is a robust repair mechanism that appears highly active in the majority of cell types,
including non-dividing cells. However, NHEJ is error-prone and can often result in the removal
or addition of between one and several hundred nucleotides at the site of the DSB, though such
modifications are typically <20nt (Jinek et al., 2012). If these small insertions/deletions (indels)
occur within an open-reading frame, there is a high likelihood that the gene will be functionally
disrupted due to frame shift or deletion of critical amino acid residues from the expressed
protein. Similarly, disruption of critical regulatory elements can significantly alter the expression
level of target genes.
Figure 4 CRISPR-Cas9-Mediated Genome Editing Strategies

CRISPR-Cas9-mediated DNA cleavage creates a double-strand break (DSB) at the target locus. The DSB can be
repaired by non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Genome Editing Strategy
Image, 2015).
CTX001 relies on DNA repair by NHEJ to create insertion or deletion (indels) of one or more
nucleotides at the site of the DSB within the BCL11A enhancer locus. When the critical regulatory
elements are modified, they can significantly alter the expression level of BCL11A. The intended
gene modification employed by CTX001 disrupts an essential transcription factor binding site
(GATA1), leading to reduced BCL11A expression and the downstream effect of HbF induction.

1.4. BCL11A
BCL11A gene encodes a transcription factor, highly expressed in the brain, B-lymphocytes, and
adult erythroid precursor lineages, that acts as repressor of human γ-globin gene and exerts this
effect by binding directly to the globin locus on chromosome 11. Sequence variants within the
BCL11A gene (located on chromosome 2) were demonstrated to correlate with HbF expression
in genome-wide association studies (Menzel et al., 2007; Lettre et al., 2008; Uda et al., 2008).
Knockdown of BCL11A in primary human erythroid precursor cells resulted in increased HbF
(Sankaran et al., 2008). Furthermore, BCL11A knockout was shown to disrupt developmental
silencing of human fetal γ-globin in transgenic mice (Sankaran et al., 2009). Conditional
knockout in adult sickle cell disease transgenic mice leads to reactivation of γ-globin and
amelioration of symptoms (Xu et al., 2011).
Haploid missense mutations in BCL11A gene are associated with persistent HbF ranging from
8.7-20.8% (Dias et al., 2016), and haploinsufficiency in BCL11A is associated with persistent
HbF ranging from 3.1-29.7%, and where measured, approximately 50% reduction in BCL11A
messenger ribonucleic acid (mRNA) (Basak et al., 2015; Funnell et al., 2015; Dias et al., 2016)
in non-thalassemic patients.
CRISPR-Cas9-mediated disruption of the BCL11A intronic erythroid enhancer in human
erythroid precursors led to a 20% increase above background in HbF expression (Canver et al.,
2015) These data form the approach of CTX001 to pursue disruption of BCL11A erythroid
enhancer as a target for CRISPR-Cas9 modification to re-express HbF in subjects with TDT.
1.5. Investigational Product: CTX001

CTX001 is a cellular product consisting of autologous CD34+ human hematopoietic stem and
progenitor cells (hHSPCs) modified by CRISPR-Cas9-mediated gene editing. The target of the
CRISPR-Cas9 gene editing is the erythroid lineage-specific enhancer region of the BCL11A
gene located on intron 2 between exons 2 and 3 on chromosome 2. More specifically, the edits
created with the highly specific guide, SPY101, target a critical transcription factor binding site
(GATA1) at an erythroid lineage-specific enhancer region (DNase I hypersensitive site +58,
DHS+58) of the BCL11A gene. SPY101-RNP introduces DSBs in DNA in a sequence-
dependent manner. Repair of the DSB by NHEJ results in DNA insertion/deletions (indels),
intended to disrupt GATA1 binding, thereby lowering BCL11A expression, with concomitant
increases in γ-globin and fetal hemoglobin levels. This leads to subsequent decrease in
ineffective erythropoiesis (due to a reduction in uncomplexed α-globin chains) and improved
erythrocyte survival (due to decrease in hemolysis), and ultimately amelioration of sequelae of
anemia and reduction in transfusion needs (Musallam et al., 2012). This well-documented
amelioration of clinical symptoms occurs in patients with β-thalassemia and HPFH and forms the
basis for the therapeutic approach of CTX001 (Figure 5).

higher dose of CD34+ edited cells and a potential dose-response relationship may exist for
efficacy.
The Sponsor, along with the Data Monitoring Committee (DMC), will monitor for any potential
dose related toxicities and in such event a maximum target dose will be set.
1.10. Risk Assessment

The Sponsor has carefully considered potential risks that may be associated with this novel
therapeutic approach of genetically editing hHSPCs using the CRISPR-Cas9 technology and
reinfusing CTX001 following standard myeloablative conditioning with busulfan. The Sponsor
views the overall risk to be acceptable considering the life-shortening nature, and lack of curative
or transformative therapies available for the majority of patients with transfusion dependent β-
thalassemia and the ensuing morbidity associated with chronic RBC transfusions.
1.10.1. Potential Risks of CTX001

CRISPR-Cas9 has the proposed advantage of cleaving DNA in a highly specific manner at the
intended cutting site, the erythroid lineage-specific enhancer region of the BCL11A gene. The
intention of selecting a highly-specific guide, such as SPY101, was to reduce the risk of
tumorigenesis, the main potential risk associated with CTX001, due to potential off-target cutting
which may result in tumor suppressor inactivation or oncogene activation. Thus, when selecting
a candidate gRNA, a comprehensive system was applied to design and test gRNAs for high on-
target editing efficiency and low numbers of off-target cleavage sites.
To assess potential risks, a non-clinical safety program was designed in accordance with the
specific requirements and recommendations for cell-based and gene therapy medicinal products
as outlined by regulatory guidance documents, taking into account the nature of the product and
its intended use (please see full description in the Investigator’s Brochure). In the non-clinical
genotoxicity and toxicology assessment of CTX001, the on-target and potential off-target editing
was extensively and systematically evaluated using multiple methods. We established that
SPY101-RNP demonstrates a high rate (88%) of on-target editing and no detectable off-target
editing at the detection level as compared to unedited cells. Karyotyping analysis did not identify
any chromosomal translocations or other detectable abnormalities. To further demonstrate lack
of tumorigenesis potential of CTX001, we conducted biodistribution and combined toxicity and
tumorigenicity studies. There was no evidence for an increased incidence of any abnormal
finding in animals receiving CTX001 compared to animals receiving control (unedited) cells.
Overall, the thorough safety and toxicological characterization of CTX001 supports first in
human testing in the proposed clinical protocol. Although the current data do not provide any
evidence for oncogenesis, the overall risk of the CTX001 treatment process, including the
potential risk of oncogenesis, is unknown. Subjects will be informed of this potential risk and
will be monitored for up to 15 years on the long-term follow-up protocol or registry.
Further details of toxicology studies performed with CTX001 pre-clinically are reported in the
accompanying Investigator’s Brochure.

1.10.2. Potential Risks of Mobilization, Apheresis and Autologous Transplantation

This first-in-human study with CTX001 in subjects with TDT takes advantage of the standard
transplantation platform at specialized transplant centers. Sites and investigators who will
participate in this trial are well-experienced in the methods of mobilization, apheresis and
infusion of cryopreserved cell products and managing of potential adverse events (AEs)
associated with these procedures and busulfan myeloablation. This level of expertise will ensure
that the potential risks to the subjects associated with these procedures will be minimized.
Complete information regarding medications listed below is contained in the prescribing
information and should be used as a reference by the investigator to inform potential subjects and
guide monitoring and risk management.
• Risk of Filgrastim
Subjects will undergo dual mobilization with filgrastim and plerixafor. Filgrastim will be
administered subcutaneously. Common AEs include headache, fever, muscle, joint and bone
pains, thrombocytopenia, dyspnea, and nausea/vomiting. Enlargement or rupture of the spleen,
loss of appetite, chest pain, and low blood pressure have been reported in <10% patients. Rare
(<1%) but serious events include thrombosis and lung toxicity.
• Risk of Plerixafor
Plerixafor will be administered subcutaneously. Common AEs include injection site reaction and
bruising, diarrhea, nausea, headache, dizziness, insomnia, fatigue, and muscle, bone and joint
pain. Uncommon side effects seen in less than 1% of patients are splenic rupture, anaphylactic
reactions, and vasovagal reactions.
During mobilization subjects will be closely monitored and frequent abdominal exams will be
performed by experienced investigators and staff.
• Risk of Apheresis
All subjects will undergo apheresis following 4 days of mobilization. Apheresis may occur for
up to 3 consecutive days in order to collect the required amount of CD34+ stem cells for CTX001
manufacturing (minimum of 15 x 106 CD34+ cells/kg) and for a backup stem cell dose
(minimum of 2 x 106 CD34+ cells/kg). Apheresis will be performed per the site’s standard
procedures. Side effects associated with apheresis are related to intravascular volume changes
and electrolyte abnormalities. Most common mild to moderate side effects include paresthesias,
lethargy, dizziness, sweating, nausea and/or vomiting, arrhythmia, hypotension, and
hypocalcemia. Severe AEs may include loss of consciousness and seizures. The trial sites are
experienced in preventing and managing these potential risks.
• Risk of Busulfan Conditioning
Known risks of busulfan conditioning include infertility, myelosuppression, infections, hepatic
veno-occlusive disease (VOD), pulmonary toxicity, seizures, alopecia, and a long-term
malignancy potential. Subjects participating in the trial will be fully informed of these risks and
offered the option of cryopreservation of oocytes for females and sperm for males to facilitate
management of infertility should it occur.

• Risk of Defibrotide
Busulfan has a known potential risk of VOD. Defibrotide will be allowed to be used
prophylactically at the discretion of investigator and in instances when VOD develops. Subjects
will be closely monitored for potential risks associated with defibrotide which may include
bleeding (including cerebral, pulmonary, gastrointestinal or genitourinary bleeding),
hypotension, nausea, vomiting, diarrhea, and infection.
2. STUDY OBJECTIVES AND ENDPOINTS
2.1. Objectives
2.1.1. Primary
• To evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
modified CD34+ HSPCs (CTX001) in subjects with TDT
2.1.2. Secondary
• To quantify percentage of edited alleles in peripheral blood leukocytes and bone
marrow cells
• To assess the production of HbF post-CTX001 infusion
• To assess the effects of infusion of CTX001 on disease-specific events and clinical
status
2.1.3. Exploratory
• To assess the ability of biomarkers to characterize CTX001 effect and predict
treatment outcomes
2.2. Endpoints
2.2.1. Safety Endpoints
Proportion of subjects with engraftment. Engraftment is defined as absolute
neutrophil count (ANC) ≥500/µL for three consecutive days. Engraftment failure is
defined as any subject not achieving neutrophil engraftment by Day +42 following
CTX001 infusion or if backup unmodified CD34+ cells are utilized. Separately,
platelet engraftment will also be analyzed.
• Time to engraftment
• Frequency and severity of collected AEs from signing of informed consent through
Month 24 visit as assessed by the National Cancer Institute (NCI) Common
Terminology Criteria for Adverse Events (CTCAE) v4.03.
• Incidence of transplant-related mortality (TRM) at 100 days and 1 year post-CTX001
infusion. TRM is defined as death possibly related to the transplantation procedure as
assessed by the investigator.
• All-cause mortality

2.2.2. Primary Efficacy Endpoint

2.2.3. Key Secondary Efficacy Endpoint
2.2.4. Secondary Endpoints
• Proportion of subjects achieving transfusion independence for at least 12 months
(TI12).
• Proportion of alleles with intended genetic modification present in peripheral blood
leukocytes over time. Intended genetic modifications are indels that modify the
sequence of the erythrocyte-specific enhancer in intron 2 of BCL11A
• Proportion of alleles with intended genetic modification present in bone marrow cells
over time
• Change in fetal hemoglobin concentration (pre-transfusion) over time
• Change in health-related quality of life (HRQoL) from baseline over time using
EuroQol Questionnaire – 5 dimensions – 5 levels of severity (EQ-5D-5L)
• Change in HRQoL from baseline over time using the Functional assessment of cancer
therapy-bone marrow transplant questionnaire (FACT-BMT)
• Change in parameters of iron overload, including:
o Liver iron concentration (LIC) and cardiac iron content (CIC) from baseline
as assessed by T2* magnetic resonance imaging (MRI)
o Change in serum ferritin level from baseline over time
• Proportion of subjects receiving iron chelation therapy over time
2.2.5. Exploratory Endpoints
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-
cells) from baseline (pre-transfusion) over time
• Expression of α-globin and non-α-globin mRNA in circulating reticulocytes over time
• Change in erythropoietin (EPO) concentrations over time
• Change in hepcidin concentrations over time
• Assessment of erythropoiesis on bone marrow analysis compared with baseline over
time

3. STUDY POPULATION
3.1. Number of Subjects

In the first part of the study, 12 adult non-β0/β0 TDT subjects will be enrolled and treated with
CTX001. Replacement subjects may be added if subjects fail screening, withdraw consent prior
to CTX001 infusion, or do not receive CTX001 infusion for any other reason.
After more subjects have been treated with CTX001, the trial may be expanded to additional
18 subjects (expansion cohort) upon review of available safety and efficacy data and agreement
from the DMC.
3.2. Subject Inclusion Criteria

Subjects must meet all the following inclusion criteria to be eligible for enrollment into the
study:
1. Age ≥18 and ≤35 years of age.
2. Able to provide written informed consent.
a. Documented homozygous β-thalassemia (with the exception of the β0/β0 genotype) or
compound heterozygous β-thalassemia including β-thalassemia/hemoglobin E (HbE).
Subjects can be enrolled based on historical data, but a confirmation of the genotype using
the study central laboratory will be required before busulfan conditioning. The β0/β0
genotypes are defined using the HbVar Database.
b. A history of at least 100 mL/kg/year or 10 units/year of packed RBC transfusions in the
prior 2 years before signing the consent.
6. Access to detailed medical records on packed RBC transfusions, including volume or units of
packed RBCs and associated pre-transfusion Hb values, and in-patient hospitalizations, for at
least the 2 years prior to consent.
7. Female subjects of childbearing potential (postmenarcheal, has an intact uterus and at least
1 ovary, and is less than 1 year postmenopausal) must agree to use acceptable method(s) of
contraception from consent through at least 6 months after CTX001 infusion.
8. Male subjects must agree to use effective contraception (including condoms) from start of
busulfan conditioning through at least 6 months after CTX001 infusion.
9. Willing to participate in an additional long-term follow-up study or registry after completion
of this study.

3.3. Subject Exclusion Criteria

Subjects meeting any of the following criteria are not eligible for enrollment in the study:
1. An available 10/10 Human Leukocyte Antigen (HLA)-matched related donor.
2. Prior allo-HSCT.
3. Subjects with associated α-thalassemia and >1 alpha chain deletion.
4. Subjects with a β0/β0 thalassemia genotype or sickle cell beta thalassemia variant
5. Clinically significant and active bacterial, viral, fungal, or parasitic infection as determined
by the investigator.
6. White blood cell (WBC) count <3 × 109/L or platelet count <50 × 109/L not related to
hypersplenism.
8. History of any illness or any clinical condition that, in the opinion of the investigator, might
confound the results of the study or pose an additional risk in administering study drug to the
subject. This may include, but is not limited to: history of relevant drug allergies; history of
cardiovascular or central nervous system disease; history or presence of clinically significant
pathology; or history of mental disease.
9. Any prior or current malignancy or myeloproliferative disorder or a significant
immunodeficiency disorder.
a. Aspartate transaminase (AST), alanine transaminase (ALT) >3 x the upper limit of
normal (ULN), or direct bilirubin value >2 x the ULN, or:
c. History of cirrhosis or any evidence of bridging fibrosis, or active hepatitis on liver
biopsy. Liver biopsy is required when LIC is ≥15 mg/g on T2* MRI of liver. If a liver
biopsy has been performed less than 6 months prior to consent, it does not need to be
repeated.
11. A cardiac T2* <10 ms by MRI or left ventricular ejection fraction (LVEF) <45% by
echocardiogram.
13. Diffusing capacity of the lungs for carbon monoxide (DLco) <50% of predicted (corrected
for hemoglobin and/or alveolar volume).
14. Prior treatment with gene therapy.
15. Intolerance or known sensitivity to plerixafor, granulocyte colony stimulating factor (G-CSF)
products (e.g., filgrastim), or busulfan. Prior anaphylaxis with excipients of CTX001 product
(Dimethyl sulfoxide [DMSO], Dextran).

16. Positive serology for human immunodeficiency virus-1 (HIV-1) or human immunodeficiency
virus-2 (HIV-2), hepatitis B virus (HBV; hepatitis B core antibody [HBcAb] and positive HBV
polymerase chain reaction [PCR]), or hepatitis C virus (HCV) (positive HCV PCR). Positive
serology for syphilis, toxoplasmosis or any other infectious disease marker as required by local
testing for cellular processing.
17. Participation in another clinical study with an investigational drug within 30 days of
screening or fewer than 5 half-lives of the investigational agent, whichever is longer from
screening.
18. An assessment by the investigator that the subject would not comply with the study
procedures outlined in the protocol.
3.4. Enrolled Subjects and Screen Failures

Enrolled subjects are defined as subjects who consent to participate in the clinical trial and
whose eligibility is confirmed (meet the inclusion/exclusion criteria).
Screen failures are defined as subjects who consent to participate in the clinical trial but do not
meet the inclusion/exclusion criteria.
Rescreening and Repetition of Screening Assessments: Individuals who do not meet the
eligibility criteria for participation in this trial upon initial screening (screen failure) can be
rescreened once. Subject should be re-screened within 90 days of initial screen failure. All
screening tests should be repeated to determine eligibility except for: T2* MRI of liver and heart
(if performed within 6 months and pulmonary function tests (PFTs) and ECHO if performed
within 3 months).
Rescreened participants will keep the same participant number assigned during the initial
screening process.
3.5. Waivers of Inclusion/Exclusion Criteria

Waivers will not be permitted in the study.
3.6. Contraception
Study participation requires a commitment from the male subject and his/her partner to use
2 highly effective methods of birth control.
Female subjects of childbearing potential, must agree to use acceptable, highly effective methods
of contraception to avoid pregnancy from consent through at least 6 months after CTX001
infusion.
Non-sterile male subjects who are or may become sexually active with female partners of
childbearing potential must agree to use acceptable, highly effective methods of contraception to
avoid fathering a child from start of busulfan conditioning through at least 6 months after
CTX001 infusion.

Acceptable methods of contraception for subjects and their partners are listed below. If
applicable, additional contraception requirements may need to be followed according to local
regulations and/or requirements.
Contraception for the couple is waived for the following:
• True abstinence for the subject, when this is in line with the preferred and usual lifestyle
of the subject. Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-
ovulation methods) and withdrawal are not acceptable methods of contraception.
• If the male is infertile (e.g., bilateral orchiectomy). Infertility may be documented
through examination of a semen specimen or by demonstration of the absence of the vas
deferens by ultrasound before mobilization.
• If the female is of non-childbearing potential, as described below.
Female Subjects
Acceptable, highly effective methods of contraception to avoid pregnancy must be used from
consent through at least 6 months after CTX001 infusion.
• Female bilateral tubal ligation performed at least 6 months previously.
• Female diaphragm, cervical cap, or vaginal sponge, each with spermicide (where
available).
• Female continuous use of an intrauterine device (non-hormone releasing or hormone
releasing) for at least 90 days before mobilization.
• Female combined (estrogen and progestogen-containing) or progestogen-only oral
hormonal contraception associated with inhibition of ovulation if successfully used for at
least 60 days before mobilization or with a second form of approved contraception for at
least 60 days after beginning hormonal contraception.
Male Subjects
Acceptable contraceptive methods must be used from mobilization through 6 months after
CTX001 infusion and include the following:
• True abstinence, when this is in line with the preferred and usual lifestyle of the subject.
Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods)
and withdrawal are not acceptable methods of contraception.
• Condom with spermicide (either as a single product if commercially available and/or
allowed according to local regulations; otherwise condom and spermicide as separate
products). Local regulations may require use of an additional acceptable method of
contraception.
• Vasectomy (with a negative sperm post-vasectomy semen analysis) at least 6 months
before start of mobilization, and 1 barrier method of contraception.
Acceptable contraceptive methods for female partners of male subjects:
• Bilateral tubal ligation performed at least 6 months previously.
• Continuous use of an intrauterine device for at least 90 days before start of mobilization

3.8. Subject Withdrawal

A subject (or legally acceptable representative) may withdraw consent to participate in the study
at any time for any reason without prejudice to his or her future medical care. If a subject
withdraws consent, the date and reason for withdrawal should be documented. Subject data
collected up to the date of consent withdrawal will be included in the analyses. If the subject
withdraws consent for the study, no further evaluations should be performed, and no additional
samples will be collected. The Sponsor may retain and continue to use any data and samples
collected before such withdrawal of consent. Wherever possible, the tests and evaluations listed
for the end of study visit should be carried out. The Sponsor should be notified of all study
withdrawals, as soon as possible.
If subject wishes to withdraw consent to participate in the study after infusion of CTX001, the
investigator should ask the subject if he/she consents to have information collected through
review of medical records or quarterly telephone interviews (partial withdrawal of consent) or
through enrollment in the long-term follow-up study or registry. All subjects who withdraw after
CTX001 infusion should be followed for safety whenever possible.
Other reasons for removal of a subject from the study prior to start of busulfan include:
• Any medical condition that, in the opinion of the investigator, would put the subject at
risk for continuing study-related procedures or follow-up.
• If a subject is found not to have met eligibility criteria or has a major protocol deviation
• Subject becomes pregnant or begins breastfeeding
• Failure to successfully manufacture the minimum dose of CTX001 or deliver CTX001 to
the site
• Administrative decision by Sponsor
If a subject chooses to withdraw from the study after the start of conditioning with busulfan and
before administration of CTX001, the subject’s stored, unedited backup stem cells will be
infused for safety reasons.
In the unlikely event that a 10/10 HLA matched related donor becomes available once the
subject had been enrolled in the trial and before starting administration of busulfan, then the
subject would be withdrawn from the trial.
3.9. Replacement of Subjects

Subjects who fail screening, withdraw consent prior to CTX001 infusion, or do not receive
CTX001 infusion for any other reason may be replaced.
4. STUDY DESIGN
4.1. Study Overview
This is a single-arm, open-label, multisite, single dose, Phase 1/2 study that will enroll subjects
18 to 35 years of age with non-β0/β0 TDT. Transfusion dependence is defined as a history of at

See Section 6 (Treatment Plan) for further description of mobilization, apheresis, busulfan
conditioning, infusion, and shipment to the manufacturing facility.
Stage 3: Myeloablative conditioning (Stage 3A), and infusion of CTX001 (Day 1, Stage 3B)
Stage 3A – Myeloablative conditioning: After the CTX001 product is received at the site and it
has been confirmed that the backup cells have been stored and are available, the subject will be
hospitalized and undergo myeloablative conditioning with busulfan.
Busulfan will be administered intravenously (IV) daily at a starting dose of 3.2 mg/kg/day (based
on weight collected 3-7 days prior to initiation of busulfan) for 4 consecutive days. Once-daily
dosing is the preferred schedule; however, the busulfan dosing regimen may be adjusted to be
given every 6 hours (Q6H) per the site’s standard practice.
The dose of busulfan will be adjusted based on the pharmacokinetics (PK) of the first busulfan
dose to maintain appropriate levels for myeloablation. The average target area under the curve
(AUC) for subjects receiving a starting dose of 3.2 mg/kg/day for 4 days is 5000 µM*min
(range: 4500 to 5500 µM*min); equivalent to a target cumulative busulfan exposure of 90 mg x
hr/L (range 80-100 mg x hr/L). The mean target AUC for subjects administered busulfan Q6H
for 4 days is 1125 µM*min (range: 900 to 1350 µM*min).
Clinical sites may use a test dose of busulfan 3-7 days before beginning myeloablation to pre-
determine busulfan dose. Prophylaxis of VOD using defibrotide may be used per investigator’s
discretion.
Stage 3B - CTX001 infusion: Infusion of CTX001 will occur at a minimum of 48 hours
following the completion of busulfan infusion and at a maximum of 7 days after the completion
of busulfan infusion.
On Day 1, after thawing, the entire dose of CTX001, which at a minimum will be 3.0 × 106
CD34+ cells/kg, will be infused through a central venous catheter. All vial(s) containing CTX001
should be infused.
Stage 4: Follow-up, through engraftment and up to 24 months after CTX001 infusion
Stage 4A - Post-infusion until hospital discharge (engraftment): Subjects will be followed daily
in the transplant unit and receive supportive care according to standard practices for subjects
undergoing hematopoietic stem cell transplant (HSCT). Subjects will be monitored for AEs and
engraftment. Packed RBC and platelet transfusions will be given to subjects per standard
practices/investigator judgment for patients undergoing HSCT. Subjects will be discharged from
the transplant unit upon neutrophil engraftment (defined as ANC ≥500/µL for 3 consecutive
days) and stabilization of major medical issues as per local hospital guidelines and/or
investigator judgment.
In the unlikely event that engraftment is not achieved within 42 days of CTX001 infusion, the
subject will receive the backup CD34+ stem cells.
Stage 4B - Post-engraftment to 2-years post-infusion: Subjects will be followed for 24 months
after CTX001 infusion with physical exams, laboratory and imaging assessments, and AE
evaluations. Subjects will be allowed to restart iron chelation approximately 1 month after
CTX001 infusion according to the site’s management guidelines and/or investigator judgment.

Following engraftment, transfusions of packed RBCs should be avoided for Hb ≥9 g/dL, unless
medically indicated (e.g., symptomatic anemia or as a requirement for surgery). It is
recommended that subjects should receive packed RBC transfusions for Hb <7.0 g/dL, while
medical judgement is advised to transfuse for Hb levels of 7-9 g/dL based on a subject’s clinical
needs.
4.2. Study Duration

The duration of Stage 1 will be approximately 1-3 months; Stage 2 approximately 2-3 months;
Stage 3 approximately 1 month; Stage 4 approximately 2 years.
Subjects will be followed on study for a total of approximately 2.5 years after signing the
consent and for 2 years after CTX001 infusion.
Additionally, all subjects infused with CTX001 product will be asked to enroll in a long-term
follow-up study or registry following completion of or withdrawal/discontinuation from this
study.
4.3. Study Enrollment Plan

The first two subjects enrolled in the study will receive CTX001 in a staggered manner. The
second subject will not start busulfan conditioning until the first subject achieves neutrophil
engraftment (ANC ≥500/µL for 3 consecutive days by Day 42) and the available engraftment
and safety data have been reviewed by the DMC. After the first subject achieves neutrophil
engraftment and the DMC has reviewed and approved dosing in the second subject, the second
subject will receive CTX001. Once the second subject achieves neutrophil engraftment and does
not experience Grade ≥3 AEs other than those typically associated with busulfan conditioning or
autologous transplant procedure, the subsequent subject(s) can undergo conditioning and
CTX001 infusion concurrently. In the event that the second subject infused with CTX001
experiences Grade 3≥AEs other than those typically associated with busulfan conditioning or
autologous transplant procedure, a DMC meeting will be convened and data will be reviewed
before the remaining subjects can undergo conditioning and CTX001 infusion. All steps that
precede busulfan myeloablation such as consent, screening, and stem cell collection may proceed
concurrently without staggering subjects.
5. STUDY STOPPING RULES

5.1. Enrollment Suspension Criteria
Enrollment in this study will be temporarily suspended for any of the following safety reasons:
• Death on study after CTX001 infusion.
• Detection of leukemia, myelodysplastic disease, or myeloproliferative disorder in a
subject who has received CTX001
• Failure in two subjects to achieve neutrophil engraftment after infusion of CTX001, as
defined by lack of engraftment by day 42 or administration of backup cells.

• Any unexpected, clinically significant, Grade 3 or 4 toxicities at least possibly related to

busulfan or CTX001 in 3 subjects.
Enrollment in this study will be temporarily suspended for lack of efficacy if:
• HbF <1g/dL at 9 months in 4 subjects and ≥80% of those enrolled
• The proportion of alleles with intended genetic modification present in peripheral blood
leukocytes is <10% in 4 subjects and ≥80% of those enrolled at 2 consecutive timepoints
after neutrophil engraftment
If there is any such event(s) of lack of safety and/or efficacy, enrollment will be temporarily
suspended, and all available data will be reviewed by the Steering Committee (SC) and the
Sponsor. After evaluation, and following a review of the data with the DMC, the SC and Sponsor
may decide to restart enrollment in the study, amend the study, or permanently suspend
enrollment and remove all subjects who have not received CTX001 from the study.
Additionally, the Sponsor may temporarily suspend enrollment at any time if the benefit-risk
assessment is suspected to be no longer acceptable. In this case, all available data will be
reviewed by the DMC. After evaluation, the DMC will make a recommendation regarding the
conduct of the study to the SC and Sponsor. Based on this recommendation, the SC and Sponsor
may decide to restart enrollment in the study, amend the study, or permanently suspend
In the event enrollment is permanently stopped, subjects who are already enrolled in the study
will not undergo stem cell mobilization, or myeloablation with busulfan, and will have an end of
study visit. Subjects who have already been treated with CTX001 will remain in the study and be
followed up or transitioned to a long-term safety follow-up study or registry.
In the unlikely event that enrollment is suspended after a subject (or subjects) has received
busulfan and before they have received CTX001, the decision to infuse CTX001 or the backup
unedited CD34+ cells to the subject will be up to the investigator’s discretion after discussion of
the potential risks with the subject. The investigator should inform the medical monitor of the
decision before infusion of cellular product.
If enrollment in the study is suspended, the Sponsor will immediately inform all appropriate
parties including Principal Investigators (PIs), Ethics Committees (EC), Institutional Review
Boards (IRB) and competent authorities.
5.2. Stopping Rules

Stopping rules for individual subjects are as follows:
risk for continuing study-related treatments or follow-up.
prior to start of busulfan conditioning
• Subject becomes pregnant or begins breastfeeding prior to start of busulfan conditioning

• Failure to successfully manufacture CTX001 or deliver CTX001 to the site after two
mobilization cycles
5.3. End of Study Definition

End of study will be defined as the date the last subject completes the 24-month follow-up
period. The Sponsor will notify all applicable regulatory agencies in accordance with local
requirements when the study has ended.
5.4. Sponsor’s Termination of Study

This study may be discontinued at any time due to safety concerns, efficacy concerns, failure to
meet expected enrollment goals, administrative reasons or at the discretion of the Sponsor.
Should the study be terminated prematurely, the Sponsor will provide written notification to all
investigators and regulatory authorities and will specify the reason(s) for early termination. The
investigator must inform the EC or IRB promptly and provide the reason(s) for the termination.
All subjects infused with CTX001 will be asked to be followed for up to 15 years after infusion
on a long-term follow-up study or registry.
6. TREATMENT PLAN
6.1. Description of CTX001
CTX001 is composed of autologous CD34+ hHSPCs modified with CRISPR-Cas9 in a
permanent fashion at the erythroid lineage-specific enhancer of the BCL11A gene. CTX001 is
suspended in a cryopreservative solution containing (CryoStor CS-5; sterile, protein-free, serum-
free cell freezing medium containing 5% DMSO, manufactured under current Good
Manufacturing Practice [cGMP]), and supplied in an infusion vial(s). Further description is
provided in the reference manual.
6.2. Mobilization and Apheresis

6.2.1. Mobilization
Prior to the start of mobilization each subject will be examined by an apheresis-experienced
physician and deemed to be appropriate to undergo mobilization and apheresis as per Table 2
and site’s standard procedures. Examples of ineligibility include hemodynamic instability,
positive infectious serologies, or active infection. If the subject is found not to be appropriate to
undergo mobilization and apheresis, they may be re-assessed within two weeks if the ineligibility
is due to an acute process that will resolve. Otherwise subjects should be withdrawn from the
study and undergo end of study visit.
Decision on whether a central line is required should also be made by the apheresis-experienced
nurse or physician. Mobilization will consist of a combination of filgrastim and plerixafor.
Filgrastim will be administered subcutaneously at a dose of 5 μg/kg/dose every 12 hours for 5-
6 days. Dose will be based on weight taken within 5 days of the first day of mobilization.

Retrospective information on packed RBC transfusions and iron chelation will be recorded from
24 months prior to date of consent study screening.
6.10.2. Venous Access

A central venous catheter will be used for apheresis if deemed to be necessary and for
administration of the conditioning regimen, infusion of CTX001, and for clinical care of the
subject following CTX001 infusion.
6.10.3. Transfusions
Prior to start of apheresis procedure and at least one month prior to planned initiation of busulfan
conditioning, subjects should be transfused to achieve goal of Hb ≥11 g/dL. This is done to
suppress ineffective erythropoiesis and allow for a more successful engraftment. During
hospitalization for busulfan conditioning and CTX001 infusion subjects should be supported
with packed RBC and platelet transfusions as per standard practices for patients undergoing
HSCT.
During the 24-month follow-up period after infusion of CTX001, it is recommended that subjects
receive packed RBCs for Hb ≤7g/dL and for clinical symptoms requiring transfusion. Reason for
all transfusions should be documented. Transfusions should be avoided for Hb≥9 g/dL, unless
considered clinically important (e.g., surgery).
All blood products will be filtered and irradiated as per hospital guidelines.
6.10.4. Iron Chelation

Chelation must be discontinued at least 7 days prior to starting myeloablative conditioning with
busulfan. Deferasirox is an exception which should be stopped at least 30 days before
conditioning (due to potential drug-drug interaction). If deferasirox is stopped, a different
chelator may be used for up to 7 days before busulfan conditioning.
Iron chelation with deferasirox, deferoxamine, or deferiprone should not be started until at least 1
month following CTX001 infusion to allow for stable hematopoietic recovery and avoid
potential myelosuppressive effects. Subjects should be evaluated regularly to determine whether
chelation is required. Chelators should be administered according to transfusion requirements
and iron overload level when choosing the initial dose, escalation and interruption rules as per
institutional guidelines.
Potential reasons for discontinuing iron chelation are:
• Serum ferritin <1000 μg/L
• LIC<7 mg/g
• Transfusion independence and able to undergo phlebotomy as an alternative to iron
chelation
Phlebotomy may be used instead of chelation for subjects with Hb persistently ≥11 g/dL and
who are transfusion independent.

6.10.5. Prohibited medications

G-CSF should not be administered following CTX001 infusion unless discussed with and agreed
to by the medical monitor. This is due to the hypothetical concern that early G-CSF
administration will preferentially drive differentiation of the edited reinfused CD34+ cells into
myeloid, not erythroid, lineage. Avoidance of G-CSF administration following CTX001 infusion
may allow for full maturation of CD34+ cells in all lineages. Subjects should be monitored
closely and if neutrophil engraftment has not occurred by Day+21 following CTX001 infusion,
the investigator may contact the medical monitor to discuss the potential initiation of G-CSF.
6.11. CTX001 Accountability

The investigator and Sponsor are responsible for accountability and traceability of CTX001.
The investigator will ensure that CTX001 is used in accordance with this protocol. Detailed
accountability records indicating CTX001 inventory at each clinical site, use by each subject,
and disposal will be maintained by the clinical sites. These records will document that subjects
were provided the CTX001 dose as specified in the protocol and should reconcile CTX001
received by the subject. The Sponsor or its designee will review CTX001 accountability at the
clinical sites on an ongoing basis during monitoring visits.
All excess material containing CTX001 will be considered hazardous waste and disposed of in
compliance with applicable regulations and standard hospital procedures.
6.12. Treatment Compliance

Treatment will be administered per protocol in the clinic by trained study personnel.
7. VISIT EVALUATION SCHEDULE

The procedures and assessments that will be conducted during this study are described in this
section in narrative form starting in Section 8, and presented in separate tables for
Screening/Stage 1 (Table 3), Autologous CD34+ stem cell collection/Stage 2 (Table 4),
Myeloablative conditioning and infusion of CTX001/Stages 3A/3B (Table 5), and Post-Infusion
through Follow-Up/Stage 4 (Table 6).

Table 3: Schedule of Events: Screening (Stage 1)

Procedure Screening
(up to 8 weeks)
Informed Consent X
Demographics X
Medical historya X
Blood for thalassemia genotypingb (central lab) X
Physical examination and performance statusc X
Height and weight X
Vital signsd X
12-Lead ECG X
Serum chemistrye (local lab) X
Coagulatione (local lab) X
Hematologye (local lab) X
Serum pregnancy test for females of childbearing potential (local lab; See Section 3.2) X
Infectious disease marker testinge (local lab) X
Urinalysise (local lab) X
Liver MRI (T2*) X
Liver biopsyf (if required per exclusion criteria. See Section 3.3) X
Cardiac MRI (T2*) X
Echocardiograph X
Lung diffusing capacity for carbon monoxide (DLco) [corrected] X
HRQoL assessment (EQ-5D-5L, FACT-BMT)g X
Adverse event assessment (continuous from ICF signing) Xh
Prior and concomitant medications (continuous from ICF signing) X
a
Medical history including blood transfusion records, chelation and hospitalizations for past 2 years will be collected, as well as thalassemia
history including genotype, age of diagnosis, age of first transfusion and age of initiation of chelation therapy (Section 8.1).
b
All subjects will be genotyped for thalassemia (alpha and HBB loci) irrespective of prior documentation of genotyping. Results from
genotyping will be required before busulfan conditioning.
c
Full physical examination including spleen assessment and performance status (Section 8.4).
d
Includes blood pressure (systolic and diastolic), temperature, pulse rate, respiration rate, and pulse oximetry.
e
See Section 8.8.
f
Liver biopsy should only be performed after all other assessments have been done to assess eligibility.
g
HRQoL assessment should be performed as the first assessment after obtaining Informed Consent.
h
Section 9.1, Table 8.

Table 4: Schedule of Events: Autologous CD34+ Stem Cell Apheresis (Stage 2)

Procedure Mobilization Days of Apheresis
After Study Day -5 to Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Eligibility Day -1a (if
Confirmation (prior needed)
to mobilization
start)a
Iron studiesb (local lab) X
Immunological testing (local lab)b X
Dyserythropoiesis marker testing (EPO (local lab) and hepcidin (central lab))c X
Allelic editing studies (central lab; % edited cells – baseline)c X
Hemoglobin fractionation (central lab)c X
HbF distribution, F-cells (central lab, flow cytometry)c X
α-globin and non-α-globin mRNA (central lab)c X
Exploratory research studies and assay development (central lab)c X
Bone marrow aspirate (local and central lab)d X
Sperm or oocyte banking, if requested by subject Xe
Infectious disease marker testing repeated Xf
Placement of central venous catheter for apheresis (if needed)g X
Hemolysis markers (local lab)b X
Eligibility for mobilization/apheresis re-confirmed X
Serum pregnancy test (females of childbearing potential only) Xh
Transfusion regimen (if required)i X
Physical examination and performance statusj X X X X
Vital signsk X X X X X X X
Weight Xl
Hematology (local lab) X X Xm Xm Xm
Serum chemistry (local lab) X Xm Xm Xm
Coagulation (local lab) X Xm Xm Xm
G-CSF administration (e.g. filgrastim) X X X X X Xn
Plerixafor administration ( ) X X Xo
CD34 count (local lab) X Xm Xm Xm
CD34+ stem cell apheresis for CTX001 and backup cells X X X
Shipment of CD34+ stem cells to central manufacturer X X
Adverse event assessment Continuous from ICF signingp
Prior and concomitant medications Continuous from ICF signing
a
Assessments may be performed over multiple days or visits.
b
See Section 8.8.
c
Samples drawn for the central lab should be performed immediately prior to a scheduled transfusion. See Section 8 9.
d
Bone marrow assessment for assessment of ineffective erythropoiesis and iron stain. Central assessment for baseline testing of prospective edits as well as exploratory biomarkers.
e
Sperm or oocyte banking can be performed any time before busulfan conditioning after eligibility is confirmed.
f
Repeat IDMs if screening collection is more than 28 days prior to the planned apheresis start date. Collection should be no fewer than 10 days prior to the planned apheresis date.

g
As per site’s standard guidelines. Subject must be hemodynamically stable and have no active infection as per investigator judgment.
h
Pregnancy test should be negative prior to starting mobilization and must be performed within 3 days prior to start of mobilization.
i
Goal hemoglobin >11 g/dL prior to mobilization start.
j
Physical exam should be performed within 5 days prior to the first day of mobilization and on every day of apheresis prior to starting the collection. Includes assessment of spleen size, and
performance status.
k
l
Weight taken within 5 days prior to the start of mobilization will be the weight used for all dose calculations.
m
Labs to be performed each morning prior to start of apheresis on Days 5-7. See Section 8.8.
n
For subjects with spleen, evening dose of G-CSF administered only if an apheresis collection is planned for Day 7.
o
Only if third day of apheresis is planned.
p

Table 5: Schedule of Events: Myeloablative Conditioning and Infusion of CTX001 (Stages

3A/3B)
Procedure Pre-Conditioning Conditioning Infusion
30 (±2) days 3 to 7 days prior to Day -5 to -2 Day 1
prior to initiation of
initiation of busulfana
busulfana
Transfusion regimenb X
Serum pregnancy test (females of childbearing Xc
potential only)
Physical examination and performance status X X X
Vital signs X X Xd
Weight X
ECG X Xe
Stop iron chelationf X
Hematology (local lab) X X Xg
Serum chemistry (local lab) X X Xg
Eligibility for myeloablation re-confirmed Xh Xi
Confirm rescue cells are cryopreserved at the site X
Confirm central line is placed X
Confirm CTX001 has been received at the site X
prior to initiating busulfan
Test dose of busulfan to determine optimal X
busulfan dose (optional)
Anti-seizure prophylaxis Xj
IV busulfan administration X
Busulfan pharmacokinetics Xk
IV Infusion of CTX001 (Section 6.8) X
Adverse event assessment Continuous from ICF Signingl
Prior and concomitant medications Continuous from ICF Signing
a
b
Maintain hemoglobin > 11 g/dL at least 30 days prior to starting busulfan.
c
Pregnancy test must be negative within 5 days prior to starting busulfan.
d
Measure vital signs prior to infusion and every 30 minutes after infusion up to 2 hours. Includes blood pressure (systolic and diastolic),
temperature, pulse rate, respiration rate, and pulse oximetry.
e
ECG performed prior to CTX001 infusion.
f
Iron chelation needs to be stopped at least 7 days prior to start of busulfan conditioning.
g
Collect labs prior to infusion on Day 1. See Section 8.8.
h
If screening and pre-conditioning visits are ≥3 months apart, repeat pulmonary function tests and echocardiograph to confirm eligibility.
i
Review most recent assessments (within 7 days prior to conditioning: CBC+ differential, serum chemistry, liver function tests, ECG,
physical examination, performance status and pregnancy test results and additional tests) as per site’s standard guidelines for
autologous stem cell transplant).
j
Initiate anti-seizure prophylaxis at least 12 hours before first dose of busulfan and continue until 24 hours after completion of the full
busulfan administration (as per local guidelines).
k
Blood sample will be collected for pharmacokinetic analysis for busulfan. If PK analysis is not available locally for daily results, perform
analysis on first and third day of dosing. See Appendix 1.
l

Table 6: Schedule of Events: Post-Infusion through Follow-Up (Stage 4)

Follow-Upa
Daily from D+30b D+60 D+90 D+120 D+160 D+180 D+270 D+360 D+540 D+720
Procedure Day 2 until M1 M2 M3 M4 M5 M6 M9 M12 M18 M24 ETFc
Discharge (±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±30d) (±30d)
Vital signsd X X X X X X X X X X X X
Hematology (local lab)e X X X X X X X X X X X X
Serum chemistry (local X X X X X X X X X X X X
lab)e
Coagulation (local lab)e Xf X X X X X X X X X X X
Hemolysis markers X X X X X X X
(local lab)e
Ferritin (local lab)e X X X X X X X X X X X
Physical examination X X X X X X X X X X X X
and performance status
Immunological testing X X X X X
(local lab)e
Dyserythropoiesis X X X X X X
marker testing (EPO
(local lab) and hepcidin
(central lab))g
Allelic editing studies X X X X X X X X X X X
(central lab; % edited
cells)g
Hemoglobin X X X X X X X X X X X
fractionation (central
lab)g
F-cell distribution X X X X X X X X X X X
(central lab)g
α-globin and non-α- X X X X X X X X X X X
globin mRNA (central
lab)g
Bone marrow aspirate X X X X
(local and central lab)h
Exploratory research X X X X X X
studies and assay
developmentg
Liver MRI (T2*) X X X
Lung diffusing capacity X X X
for carbon monoxide
(DLco) [corrected]
Cardiac MRI ( T2*) X X X
12-lead ECG X X X

HRQoL assessment X X X X X
(EQ-5D-5L, FACT-
BMT)i
Adverse event Continuous from ICF signingj
collection
Concomitant Continuous from ICF signing
medication
a
b
Month (M) is defined as 30 days. If Day 30 occurs while the subject is still hospitalized, the daily assessments required prior to discharge should be done in addition to the Day 30
assessments.
c
Early Termination of Follow-Up.
d
e
See Section 8.8.
f
Weekly until discharge.
g
Samples drawn for the central lab should be performed just prior to a scheduled transfusion if applicable. See Section 8.9.
h
Bone marrow assessment for assessment of ineffective erythropoiesis and iron stain. Central assessment for baseline testing of prospective edits as well as exploratory biomarkers.
i
HRQoL assessment should be performed as the first assessment at the visit.
j

8. STUDY ASSESSMENTS
8.1. Demographic/Medical History

Demographics (e.g., gender, age, race, ethnicity), 2 years of transfusion history (include any
simple, manual or exchange transfusions), and 2 years of hospitalization history (including
chelation therapy received) will be recorded. Thalassemia history such as genotype and age of
diagnosis will also be recorded. Past and present medical history, including targeted medical
history and additional medical history considered by the investigator to be significant will also be
recorded.
8.2. Transfusions
Any transfusion received and reason for transfusion (mL packed RBC/kg and number of
transfused units) will be documented from the time of consent through the end of the study.
Additionally, Hb concentration before transfusion will be documented.
8.3. Vital Signs and Weight/Height

Vital signs (blood pressure [systolic and diastolic], temperature, pulse rate, respiration rate, and
pulse oximetry) and subject weight (kg) and height (cm) will be measured per institutional
standards.
8.4. Physical Examination

Physical examinations will be performed by trained medical personnel and should include the
examination of general appearance, head, skin, neck (including thyroid), eyes, ears, nose, throat,
lungs, heart, abdomen (including spleen size), lymph nodes, extremities, vascular and
neurological systems and Karnofsky performance status (Appendix 2). Breast, anorectal, and
genital examinations will only be performed when medically indicated.
8.5. Electrocardiogram
Subjects should rest for at least 5 minutes before performing the 12-lead electrocardiogram
(ECG). The ECG should be performed with the subject in the supine position. The ECG will be
performed before any other procedures that may affect heart rate, such as blood draws. The ECG
measurement recorded pretreatment on Day 1 will serve as the subject’s baseline for all post-
infusion comparisons. A printout of the ECG traces will be made for safety review by the
investigator and maintained with source documentation.
Interpretation of the tracing should be made by a qualified physician and documented in the
medical record. Clinically significant abnormalities present at screening should be reported as
part of the subject's medical history.

Table 7: Summary of Clinical Laboratory Tests

Hematology Serum Chemistry Urinalysis Other blood tests
CBC with ALT Protein % edited cells (% of DNA

differential AST Blood sequences with the
including: Bilirubin (total and Nitrite intended edit)
Hemoglobin direct) Leukocyte esterase
WBC Albumin CD34+ cell count
Platelet count Alkaline phosphatase
Reticulocyte count Bicarbonate Globin assessment (HbF
Nucleated RBCs BUN orea and other Hb subtypes) At
MCV (Mean Calcium screening only:
corpuscular volume) Chloride
MCHC (Mean CK Genotyping of HBB and
corpuscular Creatinine alpha loci (central lab)
hemoglobin Glucose (fasting)
concentration) GGTP (Gamma-
MCH (Mean glutamyl transpeptidase)
corpuscular Magnesium
hemoglobin) Phosphorous If applicable:
RDW (Red blood Potassium Serum pregnancy test
cell distribution Sodium
width) Total protein
Uric acid
Infectious Pathogens Immunological
Testing Testing
HBV (core Ab, if CD4
positive then PCR)* CD8
HCV (Ab, if positive CD19
then PCR) CD16
HIV-1, HIV-2 CD56
(Antigen/Antibody IgG
immunoassay, RNA) IgD
Syphilis (RPR, if IgE
reactive FTA-ABS IgM
[Rapid plasma reagin/ IgA
fluorescent treponemal
antibody absorption
test])
Toxoplasmosis
Hemolysis testing Iron Studies Coagulation Dyserythropoiesis
testing
Haptoglobin Ferritin PT (Prothrombin time) EPO
LDH (Lactate Serum iron aPTT (Activated partial Hepcidin
dehydrogenase) Serum transferrin thromboplastin time)
receptor INR
Transferrin
TIBC (Total iron binding
capacity)
* Presence of anti-HBs alone indicates prior immunization and does not exclude a subject.

8.9. Blood for Biomarker Assessments

Blood samples will be collected according to Table 4 through Table 6 for evaluation of
biomarkers and their ability to characterize the effect of CTX001 and predict treatment
outcomes.
Blood samples will be collected to evaluate:
leukocyte DNA.
• Expression of α-globin and non-α-globin mRNA, in circulating reticulocytes.
• Protein based biomarkers, including but not limited to (1) hemoglobin fractionation and
quantitation in peripheral blood to assess bulk fetal hemoglobin levels, (2) the proportion of
circulating erythrocytes expressing fetal hemoglobin (F-cells), (3) EPO and hepcidin levels.
Detailed procedures for the collection of blood samples and additional procedures for processing
and handling samples will be provided in a separate document.
8.10. Blood for Exploratory Research Studies and Assay Development

Blood samples will be collected according to Table 4 through Table 6 for potential exploratory
research and assay development to better understand CTX001 and its impact on
hemoglobinopathies.
Samples of apheresed stem cells and CTX001 will be collected during manufacturing for
potential future genome analysis in case of development of malignancy thought to be related to
CTX001 and for exploratory research to better understand CTX001 and its impact on
β-thalassemia. Samples will not impact the final CTX001 dose of subject.
These data will be used for internal exploratory purposes and may or may not be included in the
clinical study report. Detailed procedures for the collection of blood samples and additional
procedures for processing and handling samples will be provided in a separate document.
8.11. Sperm and Oocyte Banking

Subjects will be offered the opportunity to undergo sperm banking or oocyte preservation
according to local SOPs and collection protocols. The procedure is optional and subjects who
choose to undergo the procedures will do so after eligibility is confirmed and prior to busulfan
conditioning (Stage 3A).
8.12. Biopsies
8.12.1. Liver Biopsy
For subjects with a LIC ≥15 mg/g on T2* MRI of liver at screening, a liver biopsy will be
performed, unless a prior biopsy was performed within the past 6 months. Subjects with biopsies
that show cirrhosis, any evidence of bridging fibrosis, or active hepatitis will be excluded from
the study.
At screening, liver biopsy should only be performed after all other assessments have been done
to assess eligibility.

8.12.2. Bone Marrow Aspirate

Bone marrow aspirate will be performed once eligibility is confirmed. Samples will be collected
to assess ineffective erythropoiesis and iron content at the site. Bone marrow aspirate samples
will be collected according to Table 4 through Table 6 for central assessment of baseline and
prospective allele editing frequency. Additionally, samples will be taken for exploratory research
to better understand CTX001 and hemoglobinopathies and a sample will also be collected for
CTX001.
8.13. Health-Related Quality of Life (HRQoL)

HRQoL surveys should be performed as the first assessment after obtaining Informed Consent,
as well as be completed by subjects at the beginning of a study visit prior to any assessments.
8.13.1. EQ-5D-5L
The EuroQol Questionnaire – 5 dimensions – 5 levels of severity (EQ-5D-5L) questionnaire
assesses a subject’s health status in a standardized way and consists of 2 parts: the EQ-5D
descriptive system and the EQ visual analogue scale (VAS) (Appendix 3).
The EQ-5D-5L descriptive system is comprised of the same 5 dimensions: mobility, self-care,
usual activities, pain/discomfort, anxiety/depression. Each dimension has 5 levels: no problems,
slight problems, moderate problems, severe problems, and extreme problems. The respondent is
asked to indicate his/her health state by ticking (or placing a cross) in the box against the most
appropriate statement in each of the 5 dimensions. This decision results in a 1-digit number
expressing the level selected for that dimension. The digits for 5 dimensions can be combined in
a 5-digit number describing the subject’s health state.
The EQ VAS records the subject’s self-rated health on a 100-point VAS with endpoints labelled
‘the best health you can imagine’ and ‘the worst health you can imagine.’ This information can
be used as a quantitative measure of health as judged by the individual respondents.
8.13.2. FACT-BMT
The Functional Assessment of Cancer Therapy – for subjects undergoing bone marrow
transplantation (FACT-BMT) questionnaire is a validated self-report questionnaire that includes
physical, social, family, emotional, and functional well-being (Appendix 4). The FACT-BMT
consists of the FACT-General (constitutes the core of all subscales) and treatment-specific
concerns of bone marrow transplantation.
Each statement in the FACT-BMT has a five point Likert-type response scale ranging from 0 to
4 (0 = “not at all”; 1 = “a little bit”; 2 = “somewhat”; 3 = “quite a bit”; and 4 = “very much”).
The subject is asked to circle or mark one number per line to indicate his/her response to the
statement as it applies to the past 7 days. Questionnaires are then scored, and the higher the
score, the better the QOL.

conditioning; and all AEs and SAEs from start of busulfan conditioning until 24 months after
CTX001 infusion (Table 8).
Table 8: Adverse Event (AE) and Serious Adverse Event (SAE) Recording
Serious Adverse
Study Time Period Adverse Events (AEs)
Events (SAEs)
Signing of the ICF to start of All grades of study procedure-related

busulfan conditioning AEs only (including AEs possibly
All SAEs
related to GCSF/plerixafor
administration and apheresis)
Start of busulfan conditioning to

All AEs All SAEs
Month 24 End of Study Visit
Every effort will be made by the investigator to assess the relationship of the AE, if any, to the
study drug(s). Causality will be classified using the categories presented in Table 9.
All subjects will be queried, using nonleading questions, about the occurrence of AEs at each
study visit. When possible, a constellation of signs and/or symptoms will be identified as
1 overall event or diagnosis. All AEs for enrolled subjects will be recorded in the case report
form (CRF) and source document. AEs for subjects who are screened but not subsequently
enrolled in the study will be recorded only in the subject's source documents. The following data
will be documented for each AE:
• Description of the event
• Classification of “serious” or “nonserious”
• Date of first occurrence and date of resolution (if applicable)
• Severity or Toxicity grade
• Causal relationship to study drug(s)
• Action taken
• Outcome
• Concomitant medication or other treatment given

Table 9: Classifications for AE Causality
Classification Definition
Related There is an association between the event and the administration of

investigational study drug, a plausible mechanism for the event to be related
to the investigational study drug and causes other than the investigational
study drug have been ruled out, and/or the event reappeared on re-exposure to
the investigational study drug.
Possibly related There is an association between the event and the administration of the
investigational study drug and there is a plausible mechanism for the event to
be related to investigational study drug, but there may also be alternative
etiology, such as characteristics of the subject’s clinical status or underlying
disease.
Unlikely related The event is unlikely to be related to the investigational study drug and likely
to be related to factors other than investigational study drug.
Not related The event is related to an etiology other than the investigational study drug
(the alternative etiology will be documented in the study subject's medical
record).
Severity will be assessed per the CTCAE v4.03 scale. If the CTCAE does not list a specific
event, then the general guideline provided in the CTCAE (Table 10) will be used for assessment:
Table 10: Grading Scale for Adverse Events

Grade 1 Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only;

intervention not indicated
Grade 2 Moderate; minimal, local, or noninvasive intervention indicated; limiting age-
appropriate instrumental activities of daily living (ADL)a
Grade 3 Severe or medically significant but not immediately life-threatening; hospitalization of
prolongation of hospitalization indicated; disabling; limiting self-care ADLb
Grade 4 Life-threatening consequences; urgent intervention indicated
Grade 5 Death related to AE
Note: A semi-colon indicates ‘or’ within the description of the grade.
a
Instrumental ADL refers to preparing meals, shopping for groceries or clothes, using the
telephone, managing money, etc.
b
Self-care ADL refers to bathing, dressing and undressing, feeding self, using the toilet,
taking medications, and not bedridden

An AE will be followed until the investigator has determined and provided the final outcome.
The outcome will be classified according to the categories shown in Table 11.
Table 11: Classifications for Outcome of an AE
Recovered/Resolved Resolution of an AE with no residual signs or symptoms
Recovered/Resolved Resolution of an AE with residual signs or symptoms

with Sequelae
Not Recovered/Not Either incomplete improvement or no improvement of an AE, such that it

Resolved (Continuing) remains ongoing
Fatal Outcome of an AE is death. “Fatal” will be used when death is at least

possibly related to the AE.
Unknown Outcome of an AE is not known (e.g., a subject lost to follow-up)
9.1.1.4. Serious Adverse Events (SAEs)

An SAE is any AE occurring during any study phase from signing of ICF that meets any of the
following outcomes:
• Fatal (death, regardless of cause, that occurs during participation in the study or occurs
after participation in the study and is suspected of being a delayed toxicity due to
administration of the study drug)
• Life-threatening, such that the subject was at immediate risk of death from the reaction as
it occurred
• In-patient hospitalization or prolongation of hospitalization. Please note that hospital
admissions during the conduct of the study that are per protocol (such as for
mobilization/apheresis, central line placement, busulfan conditioning, CTX001 infusion)
do not meet this criterion.
• Persistent or significant disability/incapacity (disability is defined as a substantial
disruption of a person's ability to conduct normal life functions)
• Congenital anomaly or birth defect
• Important medical event that, based upon appropriate medical judgement, may jeopardize
the subject or may require medical or surgical intervention to prevent 1 of the outcomes
listed above
• Engraftment failure: failure to reach ANC ≥ 500 cells/µL on three consecutive days by
Day 42 after CTX001 infusion or need to receive backup stem cells at any time during
period of neutropenia.
• Development of new malignancy.

If a subject has a hospitalization or procedure (e.g., surgery) for an event or condition that
occurred before the subject signed the ICF, and the hospitalization or procedure was planned
before the subject signed the ICF, the hospitalization or procedure will not be considered to
indicate an SAE, unless an AE caused the hospitalization or procedure to be rescheduled sooner
or to be prolonged relative to what was planned. In addition, hospitalizations clearly not
associated with an AE will not be considered to indicate an SAE.
Clarification will be made between the terms “serious” and “severe” because they are not
synonymous. The term “severe” is often used to describe the intensity (severity) of a specific
event, as in mild, moderate, or severe myocardial infarction. The event itself, however, may be
of relatively minor medical significance, such as a severe headache. This is not the same as
“serious,” which is based on subject/event outcome or action described above, and is usually
associated with events that pose a threat to a subject's life or functioning. Seriousness, not
severity, serves as a guide for defining expedited regulatory reporting obligations.
9.1.1.5. Collection of Serious Adverse Events

All SAEs that occur after obtaining informed consent and through the 24-Month Visit, regardless
of causality, will be reported by the investigator to Vertex Global Patient Safety (GPS). In
addition, all SAEs that occur after the 24-Month Visit and are considered related to study drug(s)
will be reported to Vertex GPS within 24 hours.
9.1.2. Reporting Serious Adverse Events

SAEs will be recorded on the Vertex Organized Safety Information Collection Form (hereafter
referred to as the “SAE Form”) using a recognized medical term or diagnosis that accurately
reflects the event. SAEs will be assessed by the investigator for relationship to the investigational
study drug(s) and possible etiologies. On the SAE Form, relationship to study drug(s) will be
assessed only as related (includes possibly related) or not related, and severity assessment will
not be required. For the purposes of study analysis, if the event has not resolved at the end of the
study reporting period, it will be documented as ongoing. For purposes of regulatory safety
monitoring, the investigator is required to follow the event to resolution and report to Vertex the
outcome of the event using the SAE Form.
The investigator is responsible for notifying the Vertex GPS within 24 hours of identifying an
SAE, regardless of the presumed relationship to the investigational study drug. The SAE Form
will be completed for new/initial events as well as to report follow-up information on previously
reported events. Investigators are asked to report follow-up information as soon as it becomes
available to ensure timely reporting to health authorities.
The investigator must complete, sign and date the SAE Form, verify the accuracy of the
information recorded on the SAE pages with the corresponding source documents, and send a
copy of the form and any supporting documentation (which includes laboratory data, hospital
records, and the results of relevant tests) to Vertex GPS via:
Email: globalpatientsafety@vrtx.com (preferred choice)
Fax: +1-617-341-6159
For questions, contact telephone: +1-617-341-6677

9.1.3. Expedited Reporting and Investigator Safety Letters

Vertex GPS is responsible for reporting suspected, unexpected, serious adverse reactions
(SUSARs) involving the study drug(s) to all regulatory authorities and participating investigators
in accordance with ICH Guidelines and/or local regulatory requirements, as applicable. In
addition, Vertex GPS or authorized designee, will be responsible for the submission of safety
letters to central ECs.
It is the responsibility of the investigator or designee to promptly notify the local IRB/local EC
of all unexpected serious adverse drug reactions involving risk to human subjects. Investigators
will also be notified of all unexpected, serious, drug-related events (7/15 Day Safety Reports)
that occur during the clinical trial. Each site is responsible for notifying its IRB or EC of these
additional SAEs.
9.2. Pregnancy
Subjects will be counseled to inform the investigator of any pregnancy that occurs during study.
If a subject becomes pregnant during the study, study interventions that may put the fetus at risk
will be stopped immediately.
Pregnancies (both those of female subjects and female partners of male subjects) must be
reported to the medical monitor and Vertex GPS within 24 hours of the site’s knowledge using
the Pregnancy Information Collection Form.
All pregnancies in subjects who have entered Stage 2 of the protocol will be followed through to
outcome, and the infant will be followed for 1 year after the birth, provided informed consent is
obtained. A separate ICF will be provided to explain these follow-up- activities. Birth outcomes
must be reported to the Vertex GPS using the Pregnancy Information Collection Form, even if
the subject was discontinued from the study.
Pregnancies themselves are not considered AEs or SAEs. However, any AEs or SAEs occurring
during pregnancy are to be reported following AE and SAE reporting guidelines.
All reports of congenital abnormalities/birth defects are SAEs. Spontaneous miscarriages should
also be reported and handled as SAEs. Elective abortions without complications should not be
handled as AEs.
9.3. Data Monitoring Committee

An independent DMC comprised of members with appropriate scientific and medical expertise
to monitor the study will be convened. The DMC will be charged with safeguarding the interests
of the subjects. The DMC may recommend that the Sponsor suspend enrollment, amend the
study, or discontinue the study at any time.
The Sponsor or designee will be responsible for promptly alerting the DMC regarding any
unexpected, serious AEs.
Details of the DMC structure and function, frequency of meetings, and data planned for review
will be included in the DMC charter. The DMC charter will be finalized prior to the first DMC
review meeting.

10. STATISTICAL AND ANALYTICAL PLANS

This section presents a summary of the planned analyses for this protocol. Statistical analysis
details will be provided in the Statistical Analysis Plan (SAP), which will be finalized before the
clinical data lock for the study.
10.1. Sample Size Estimation

The sample size for the first part of this study is not based on formal hypothesis testing. The
initial 12 subjects will provide a preliminary estimate of safety and efficacy of CTX001 on the
primary and the key secondary endpoints. For the primary and the key secondary efficacy
endpoints, i.e., TR and TI, which are binary outcomes, the proportions of response (TR or TI)
and the corresponding exact 95% confidence intervals will be generated.
10.2. Analysis Set

• The enrolled set includes all enrolled subjects who signed informed consent and meet the
inclusion/exclusion criteria.
• The full analysis set (FAS) is a subset of the enrolled set that includes subjects who
receive CTX001 infusion. The efficacy analyses will be performed based on the FAS.
Demographics and baseline characteristics will be summarized based on FAS as well.
• The safety analysis set is a subset of the enrolled set that includes subjects who start the
mobilization regimen. Analyses of the safety assessments will be based on the safety
analysis set. Collected AEs (including but not limited to all study procedure-related AEs
and all SAEs from the signing of the ICF to start of busulfan conditioning; and all AEs
and all SAEs from start of busulfan conditioning through the Month 24 Visit), labs, vital
signs, ECG assessments will be summarized.
10.3. General Considerations

Continuous variables will be summarized using the following descriptive summary statistics:
the number of subjects (n), mean, SD, median (Q1, Q3), minimum value (min), and maximum
value (max). The precision of the measurement for each continuous variable will be specified in
the SAP.
Categorical variables will be summarized using counts and percentages. Percentages will be
presented to 1 decimal place.
Uncertainty of estimates will be assessed by confidence intervals. All subject data will be
presented in the subject data listings; listings will display all subjects in the enrolled population,
regardless of whether or not they received study drug. Longitudinal data will be presented by
appropriate time intervals, such as monthly or quarterly depending on the nature of the data.
All individual subject data for all Subject Sets will be presented in data listings.
Baseline value, unless otherwise specified, will be defined as the most recent non-missing
measurement (scheduled or unscheduled) collected during screening and prior to start of
mobilization.

• Change in parameters of iron overload, including in LIC and CIC from baseline as
assessed by T2* MRI, and change in serum ferritin level from baseline over time
o Change in serum ferritin level as compared with baseline will be summarized as a
continuous variable over time.
o Change in LIC at Months 12 and 24 compared with baseline will be summarized
as a continuous variable over time.
o Change in Cardiac T2* at Months 12 and 24 compared with baseline will be
summarized as a continuous variable over time.
10.7. Safety Analysis

AEs will be coded according to Medical Dictionary for Regulatory Activities (MedDRA).
Analysis will include:
• Proportion of subjects with engraftment
• Time to engraftment
• Frequency and severity of AEs from signing of informed consent through Month 24 visit
as assessed by CTCAE v4.03.
• Incidence of TRM at 100 days and 1 year post CTX001 infusion. TRM defined as death
possibly related to the transplantation procedure as assessed by the investigator.
The number and percentage of subjects with AEs and treatment-emergent AEs will be
summarized by severity and seriousness in a tabular fashion according to the following
timepoints: ICF signed to start of busulfan conditioning and busulfan conditioning to 24 months
after CTX001 infusion.
The number and percentage of subjects achieving successful engraftment, together with the 95%
confidence interval will be provided.
Time to neutrophil engraftment, defined as the first of 3 consecutive days with ANC ≥500/μL
from transplantation, will be analyzed by the Kaplan-Meier method. Engraftment failure is
defined as not achieving neutrophil engraftment by Day +42 post CTX001 infusion or receipt of
backup stem cells. The number and percentage of subjects with engraftment failure will be
summarized.
Time to platelet engraftment is defined as first three consecutive days with platelet ≥20,000/ μL
without a transfusion in the past 7 days will be assessed by the Kaplan-Meier method.
Laboratory abnormalities (values outside of normal ranges, and by CTCAE grade), will also be
tabulated.
For all-cause mortality Kaplan-Meier method will be used.
The number and percentage of subjects with TRM will be summarized at 100 days and 1 year
post CTX001 infusion TRM, where TRM is defined as death at least possibly related to the
transplantation procedure as assessed by the investigator. Relatedness between SAEs leading to
death and transplantation will be as assessed by the investigators. If an SAE is assessed as being

at least possibly related to the transplantation procedure, the death will be classified as
transplant-related.
10.7.1. Adverse Events

AEs will be classified as pre-busulfan AEs (only procedure-related), and AEs from busulfan
conditioning to 24 months post-CTX001 infusion.
Details for imputing missing or partial start dates of AEs are described in the SAP.
AEs summarized by different period include the following:
• AEs by strongest relationship
• AEs by maximum severity
• AEs leading to treatment discontinuation
• Serious AEs
• AEs leading to death
Summaries will be presented by Medical Dictionary for Regulatory Activities (MedDRA)
System Organ Class and Preferred Term using frequency counts and percentages (i.e., number
and percentage of subjects with an event). When summarizing the number and percentage of
subjects with an event, subjects with multiple occurrences of the same AE or a continuing AE
will be counted once. Only the maximum severity level will be presented in the severity
summaries, and the strongest relationship level will be presented in the relationship summaries.
10.7.2. Clinical Laboratory Assessments

For laboratory measurements, the raw values and change from baseline values of the continuous
hematology and chemistry results, including coagulation studies will be summarized in
International System of Units (SI) by visit. Laboratory values will be analyzed based on specific
AE period.
In addition, a listing containing individual subject hematology, chemistry, and coagulation values
outside the normal reference ranges will be provided. This listing will include data from both
scheduled and unscheduled visits.
10.7.3. Electrocardiogram
For post-baseline ECG measurements, a summary of raw values and change from baseline values
will be provided visit for the following standard 12-lead ECG measurements: RR (msec), HR
(beats per minute [bpm]), PR (msec), QRS duration (msec), QT (msec), and QT corrected for
HR intervals (QTcF [msec]). ECGs will be summarized per the AE period.
10.7.4. Vital Signs

For vital signs measurements, the raw values and change from baseline values will be
summarized by visit: systolic and diastolic blood pressure (mm Hg), body temperature (°C),
heart rate (bpm), and respiratory rate (breaths per minute).

10.7.5. Physical Examination

PE findings will be presented as an individual subject data listing only.
10.8. Exploratory Analysis

Descriptive statistics will be used to describe the following parameters (unless stated otherwise).
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells)
from baseline (pre-transfusion) at Months 2, 3, 4, 5, 6, 9, 12, 18, 24
• Expression of α-globin and non-α-globin mRNA in circulating reticulocytes at Months 2,
3, 4, 5, 6, 9, 12, 18, 24
• Change in EPO concentrations at Months 3, 6, 12, 24
• Change in hepcidin concentrations at Months 3, 6, 12, 24
• Assessment of erythropoiesis on bone marrow analysis compared with baseline over time
• Correlations of response markers (transfusions, total and fetal hemoglobin, reduction in
ineffective erythropoiesis) with pre-treatment variables (e.g., α/non-α globin ratio, subject
genotype), cell dose and % edited cells in final product
10.9. Procedures for Handling Missing Data

In general, no missing data will be imputed, unless specified otherwise. All data will be
evaluated as observed.
A line listing of subjects who withdraw will be generated with the reason for withdrawal
including death, AE, withdrawal of consent, and lost to follow-up.
10.10. Steering Committee

A Steering Committee (SC) will be comprised of a select number of study investigators and the
Sponsor’s medical monitor(s). The SC will provide oversight of the trial, it will monitor the trial
towards its overall objectives, review relevant information as it becomes available, provide
recommendations to the Sponsor, and review DMC’s recommendations. The SC may
recommend that the Sponsor amend the protocol, or temporarily suspend the study enrollment, or
terminate the study at any time.
Details of the SC structure and function, frequency of meetings, and data planned for review will
be included in the SC charter, which will be finalized prior to the first meeting.
10.11. Endpoint Adjudication Committee (EAC)

The Endpoint Adjudication Committee (EAC) will be composed of an independent, external
group of experts with appropriate scientific background to evaluate packed RBC transfusion
needs and baseline transfusion history. This committee will be responsible for evaluating the trial
data for the two main efficacy endpoints – TR and TI. The committee will convene at the time of
interim or final analysis or at any point when the Sponsor requests an analysis to occur. The
EAC will compare the subjects’ RBC transfusion needs post-CTX001 infusion with the baseline.
Baseline for annualized RBC transfusion rate will be defined as the average of two years prior to

A CRF will be completed for each consented study subject. It is the investigator's responsibility
to ensure the accuracy, completeness, clarity, and timeliness of the data reported in the subject's
CRF. Source documentation supporting the CRF data will indicate the subject's participation in
the study and will document the dates and details of study procedures, AEs, other observations,
and subject status.
The investigator, or designated representative, will complete the CRF as soon as possible after
information is collected.
The audit trail entry will show the user's identification information and the date and time of any
correction. The investigator will provide formal approval of all the information in the CRFs,
including any changes made to the CRFs, to endorse the final submitted data for the subjects for
whom the investigator is responsible.
The Sponsor or designee will retain the CRF data and corresponding audit trails. A copy of the
final archival CRF in the form of a CD or other electronic media will be placed in the
investigator's study file.
11.3. Audits and Inspections

Authorized representatives of the Sponsor, a regulatory authority, an EC or an IRB may visit the
site to perform audits or inspections, including source data verification. The purpose of a
CRISPR audit or inspection is to systematically and independently examine all study-related
activities and documents to determine whether these activities were conducted, and data were
recorded, analyzed, and accurately reported per the protocol, GCP guidelines of the ICH, and
any applicable regulatory requirements. The investigator should contact CRISPR immediately if
contacted by a regulatory agency about an inspection.
12. QUALITY CONTROL AND QUALITY ASSURANCE

To ensure compliance with GCPs and all applicable regulatory requirements, the Sponsor may
conduct a quality assurance audit. Please see Section 11.3 for more details regarding the audit
process.
13. ETHICAL AND REGULATORY CONSIDERATIONS
13.1. Ethics Committee (EC) / Institutional Review Board (IRB)

The PI must obtain EC or IRB approval for the investigation prior to any study procedures,
including screening, are performed. Initial EC or IRB approval, and all materials approved by the
EC or IRB for this study including the subject ICF and recruitment materials must be maintained
by the investigator and made available for inspection.
13.2. Ethics Review

The final study protocol, including the final version of the ICF, must be approved or given a
favorable opinion in writing by an EC or IRB as appropriate. The investigator must submit

written approval to the Sponsor before he or she can perform any study related procedures on
subjects.
The PI is responsible for informing the EC or IRB of any amendment to the protocol in
accordance with local requirements. In addition, the EC or IRB must approve all advertising used
to recruit subjects for the study. The protocol must be re-approved by the EC or IRB upon receipt
of amendments and annually, as local regulations require.
The PI is also responsible for providing the EC or IRB with reports of any reportable serious
adverse drug reactions from any other study conducted with the investigational product. The
sponsor will provide this information to the PI.
13.3. Conduct of the Study

The study will be performed in accordance with ethical principles that have their origin in the
Declaration of Helsinki and are consistent with ICH/GCP and applicable regulatory requirements
13.4. Written Informed Consent

The PI(s) at each center will ensure that the subject is given full and adequate oral and written
information about the nature, purpose, possible risk and benefit of the study. Subjects must also
be notified that they are free to discontinue from the study at any time. The subject should be
given the opportunity to ask questions and allowed time to consider the information provided.
The subject’s signed and dated ICF must be obtained before conducting any study procedures.
The PI(s) must maintain the original, signed ICF. A copy of the signed ICF must be given to the
subject.
Whenever important new information becomes available that may be relevant to the subject’s
consent, the written ICF and any other written information provided to subjects will be revised
by the Sponsor or designee and be submitted again to the IRB/EC for review and favorable
opinion. The agreed upon, revised information will be provided to each subject in the study for
signing and dating. The investigator will explain the changes to the previous version.
14. DATA HANDLING AND RECORDKEEPING
14.1. Inspection of Records

The Sponsor or designee will be allowed to conduct site visits to the investigation facilities for
monitoring any aspect of the study. The investigator agrees to allow the monitor to inspect the
product storage area, hospital pharmacy product accountability records, subject charts and study
source documents, and other records relative to study conduct.
14.2. Retention of Records

The PI must maintain all documentation relating to the study for a period of 2 years after the last
marketing application approval, or if not approved 2 years following the discontinuance of the
test article for investigation. If it becomes necessary for the Sponsor or the Regulatory Authority
to review any documentation relating to the study, the investigator must permit access to such

records. If maintaining this documentation is no longer possible at the site, the investigator must
notify the Sponsor.
14.3. Subject Privacy and Confidentiality

To maintain subject confidentiality and to comply with applicable data protection and privacy
laws and regulations, all data provided to the Sponsor or designee, study reports, and
communications relating to the study will identify subjects by assigned subject numbers and
access to subject names linked to such numbers shall be limited to the site and the study
physician and shall not be disclosed to the Sponsor or designee. As required by applicable laws
and regulations in the countries in which the study is being conducted, the investigator will allow
the Sponsor and/or its representatives access to all pertinent medical records to allow for the
verification of data gathered and the review of the data collection process. The regulatory
authorities in other jurisdictions, including the EC/IRB, may also request access to all study
records, including source documentation, for inspection.

15. PUBLICATIONS AND CLINICAL STUDY REPORT
15.1. Publication of Study Results

Any and all scientific, commercial, and technical information disclosed by the Sponsor in this
protocol or elsewhere will be considered the confidential and proprietary property of the
Sponsor. The investigator shall hold such information in confidence and shall not disclose the
information to any third party except to such of the investigator's employees and staff as have
been made aware that the information is confidential and who are bound to treat it as such and to
whom disclosure is necessary to evaluate that information. The investigator shall not use such
information for any purpose other than determining mutual interest in performing the study and,
if the parties decide to proceed with the study, for the purpose of conducting the study.
The investigator understands that the information developed from this clinical study will be used
by the Sponsor in connection with the development of the study drug and other drugs and
diagnostics, and therefore may be disclosed as required to other clinical investigators, business
partners and associates, health authorities in different countries, and other government agencies.
The investigator also understands that, to allow for the use of the information derived from the
clinical study, the investigator has the obligation to provide the Sponsor with complete test
results and all data developed in the study.
No publication or disclosure of study results will be permitted except under the terms and
conditions of written agreement between the Sponsor and the investigator and/or the
investigator's institution.
15.2. Clinical Study Report

After completion of the study, a clinical study report, written by the Sponsor or designee in
accordance with the ICH E3 Guideline, will be submitted in accordance with local regulations.

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993-996.

17. APPENDICES

APPENDIX 1. BUSULFAN PK COLLECTION

Samples for PK analysis of busulfan will be drawn on the first and third days of dosing using
recommended guidelines below.
Recommendations for once daily dosing: Samples for busulfan PK analysis should be collected
at the following time points after the start of the infusion:
End of the 3-hour infusion (3-hours), (±5 minutes)
3 hours 15 minutes (±5 minutes)
4 hours (±15 minutes)
Recommendations for dosing Q6H: Samples for busulfan PK analysis should be collected at the
following time points after the start of the first infusion of the day:
End of the first 2-hour infusion (2 hours) (± 5 minutes)
2 hours 15 minutes (± 5 minutes)
3 hours (± 15 minutes)
Note: Samples should not be drawn from the lumen used to infuse busulfan. In both dosing
schemes, the actual time of PK collection must be recorded, not just the nominal times provided
above.
Clinical sites must either measure busulfan PK on the first and third days of administration, or
alternatively, use a busulfan test dose prior to myloablation to pre-determine the dose, with
confirmatory PK measurements on the first day of dosing. Based on the available busulfan PK
results, busulfan dose adjustments should be made for subsequent dosing. As part of this
protocol, sites are permitted to perform daily busulfan monitoring, with subsequent dose
adjustments, if that is routine practice. Specific details of busulfan dosing, PK, and dose
adjustments will be included in the eCRF.

APPENDIX 2. KARNOFSKY PERFORMANCE STATUS SCALE

APPENDIX 3. EQ-5D-5L QUESTIONNAIRE
Health Questionnaire
English version for the USA



Version 1.0 85
APPENDIX 4. FACT-BMT QUESTIONNAIRE


Vertex Pharmaceuticals Incorporated Clinical Study Protocol
CLINICAL STUDY CTX001-111

A PHASE 1/2 STUDY OF THE SAFETY AND EFFICACY OF A SINGLE
DOSE OF AUTOLOGOUS CRISPR-CAS9 MODIFIED CD34+ HUMAN
HEMATOPOIETIC STEM AND PROGENITOR CELLS (hHSPCS) IN
SUBJECTS WITH TRANSFUSION-DEPENDENT β-THALASSEMIA
Sponsor: Vertex Pharmaceuticals Incorporated
50 Northern Avenue
Boston, MA 02210, USA
Protocol Number: CTX001-111
EudraCT Number 2017-003351-38
Study Phase: 1/2
Sponsor Contact:
CRISPR Therapeutics
610 Main Street
Cambridge, MA 02139
+1 617-307-7492
Medical Safety Monitor:
Version and Date: Version 5.0, 04 February 2020
Confidentiality Statement
The information in this document contains commercial information and trade secrets that are
privileged or confidential and may not be disclosed unless such disclosure is required by applicable
laws and regulations. In any event, persons to whom the information is disclosed must be informed
that the information is privileged or confidential and may not be further disclosed by them. These
restrictions on disclosure will apply equally to all future information supplied to you that is
indicated as privileged or confidential.
Version 5.0 1
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PROTOCOL APPROVAL SIGNATURE PAGE
and Progenitor Cells (hHSPCs) in subjects with Transfusion-Dependent
β-Thalassemia
Date: 04 February 2020

Version: 5.0
Reviewed and Approved by:
Date
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PROTOCOL ACCEPTANCE FORM
and Progenitor Cells (hHSPCs) in Subjects with Transfusion-Dependent
β-Thalassemia
Date: 04 February 2020
Version: 5.0
I have carefully read this protocol and agree that it contains all of the necessary information
required to conduct this study. I agree to conduct this study as described and according to the
Declaration of Helsinki, International Conference on Harmonisation (ICH) Guidelines for Good
Clinical Practice (GCP), and all applicable regulatory requirements.
Investigator’s Signature Date
Name (printed)
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Summary of Changes to the Protocol

The previous version of this protocol (Version 4.0, 19 August 2019) was amended to create the
current version (Version 5.0, 04 February 2020). The protocol history is below.
Protocol History
Version and Date of Protocol Comments
Version 1.0, 22 November 2017 Original version
Version 1.1 (DEU), 21 May 2018 German-specific version
Version 1.2 (DEU), 27 July 2018 German-specific version
Version 2.0, 15 February 2018 Global version

Version 3.0, 16 December 2018 Global version (except Canada)
Version 3.1 (CAN), 21 February Canadian-specific version
2019
Version 4.1 (CAN), 19 August 2019 Canadian-specific version
Version 4.0, 19 August 2019 Global version (except Canada)
Version 5.0, 04 February 2020 Current global version (except Canada)
Key changes made in the current version of the protocol are summarized below.
Change and Rationale Affected Sections
Added expansion of the study to include subjects 12 to Synopsis, Section 1.8, Section 3.1, Section 3.2,
<18 years of age after DMC review of of Section 4.1, Section 4.3, Section 9.3
efficacy and safety data following infusion of CTX001 from at
least subjects ≥18 years old.
Added that approximately adult subjects will be dosed with Section 3.1, Section 4.1, Section 4.3
CTX001 before the conditioning and dosing of the first
pediatric subject.
Updated enrollment suspension criteria and individual stopping Section 5.1, Section 5.2
rules to include failure to manufacture the target cell dose of
CTX001 instead of failure to collect the target number of
CD34+ cells.
Due to the addition of subjects <18 years old, specified that for Synopsis, Section 4.1, Section 6.7
subjects less than 34 kg, institutional and/or regional practice
for busulfan dosing may be used.
Clarified that the DMC recommendation to expand the study to Synopsis, Section 4.3
dose up to 45 subjects will be done after at least subjects
have been dosed with CTX001 and the DMC has reviewed
available data.
Added the option for signing assent (where applicable) because Synopsis, Section 3.2, Section 3.7,
pediatric subjects are included in the study. Section 6.11.1, Section 9.1.1.1, Section 9.1.1.5,
Section 9.1.2, Section 9.2, Section 13.4,
Table 3 through Table 6
As the Karnofsky Performance Status Scale is used only for Synopsis, Section 3.2, Table 3, Table 6,
subjects ≥16 years old, the Lansky Performance Status Scale Section 8.4, Appendix 2
was added for use in subjects <16 years old.
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Updated the inclusion criteria and contraception requirements Synopsis, Section 3.2, Section 3.6
to specify that they apply to subjects of reproductive capacity
and who are sexually active; added that subjects whose
contraception waiver status changes during their participation
in the study will be required to follow the contraception
requirements.
Updated medical history collection to specify the collected Table 3
genotypes as alpha and HBB loci and associated traits (e.g.,
Xmn1)
Specified that the bone marrow assessment of ineffective Table 4, Table 6, Table 7
erythropoiesis includes myeloid:erythroid ratio
Added erythropoiesis markers, soluble transferrin receptor and Table 4, Table 6, Table 7
erythropoietin, to clinical laboratory tests
Added youth and parent proxy versions of EQ-5D for subjects Synopsis, Table 3, Table 6, Section 8.14.1,
<18 years old Section 2.2.4
Added Wechsler Abbreviated Scale of Intelligence for subjects Table 3, Section 8.15
12 to <18 years old at the time of assessment because there is
not a validated youth version of the Mini-Mental State
Examination
Added Pediatric Quality of Life Inventory assessment because Synopsis, Table 3, Table 6, Section 8.14.2,
a pediatric version of the FACT-BMT does not exist and Section 8.14.3, Section 2.2.4
clarified that the FACT-BMT assessment is for adults only.
Added an allowance for anesthesia during imaging studies per Section 8.7
site standard of care and subject age
Clarified the procedure for collecting stem cells for Section 4.1
manufacturing
Updated statements about current treatment options to reflect Section 1.2, Section 1.10.2
the availability of a new medicinal product, betibelogene
autotemcel (Zynteglo®)
Updated the therapeutic rationale to include pediatric subjects Section 1.8
Added language for pediatric subjects to the section about Section 1.10.2
potential risks of mobilization, apheresis and autologous
transplantation
In exclusion criterion 10, the upper limit for direct bilirubin Synopsis and Section 3.3
was changed from 2 x ULN to 2.5 x ULN. The original limit
was more stringent than required, because the criterion also
assesses other measures that are collectively more robust
assessments of advanced liver disease than direct bilirubin
alone.
Clarified that subjects will be removed from the study if they Section 3.8 and Section 5.2
have an important protocol deviation before the start of
busulfan conditioning that, in the opinion of the investigator,
would put the subject at risk for continuing study-related
procedures or follow-up.
Clarified that all prior medications taken within 30 days before Section 6.11.1
signing of the ICF will be recorded.
Added height collection for subjects <18 years old at Screening Table 4
Added height and weight collection for subjects <18 years old Table 6
at Screening during follow-up (Stage 4)
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If a subject has not engrafted at the M1 visit, the allelic editing Table 6
assessment should be collected after engraftment and before
the M2 visit.
Added the option for gonadal tissue banking, if appropriate per Synopsis, Table 3, Table 4, Section 1.10.2,
subject age and local site practice Section 3.6, Section 4.1, Section 8.12
Added that AE collection will exclude AEs possibly related to Table 8
fertility preservation procedures
Noted that subgroup analyses, such as descriptive summaries Section 10.3
of the key efficacy and safety endpoints, will be provided as
appropriate by age group and genotypes. Details will be
described in the SAP.
Additional typographical and administrative changes were also made to improve the clarity and
integrity of the document.
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SYNOPSIS
Name of Sponsor/Company:
Vertex Pharmaceuticals Inc.
Name of Investigational Product:
CTX001
Title of Study:
CTX001-111
A Phase 1/2 Study of the Safety and Efficacy of a Single Dose of Autologous CRISPR-Cas9 Modified CD34+
Human Hematopoietic Stem and Progenitor Cells (hHSPCs) in Subjects with Transfusion-Dependent
β-Thalassemia
Number of Subjects:
up to 45
Study Period: Phase of Development:
The duration of Stage 1 will be 1/2
approximately 1 to 3 months; Stage 2
approximately 2 to 3 months; Stage 3
approximately 1 month; Stage 4
approximately 2 years.
Study Rationale:
The purpose of the study is to evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
modified CD34+ Human Hematopoietic Stem and Progenitor Cells (hHSPCs) in subjects 12 to 35 years of age
(inclusive at time of informed consent) with transfusion-dependent -thalassemia (TDT). CRISPR-Cas9 editing
of autologous CD34+ hHSPCs at erythroid lineage specific enhancer of BCL11A is intended to disrupt BCL11A
gene expression in erythroid cells and consequently increase γ-globin expression in subjects with TDT. The
increase in γ-globin is expected to recreate a state similar to the elevated γ-globin and fetal hemoglobin (HbF)
observed in people with Hereditary Persistence of Fetal Hemoglobin (HPFH) to ameliorate the symptoms and
signs of TDT.
Study Objectives:
Primary
 To evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9 modified CD34+
hHSPCs (CTX001) in subjects with TDT
Secondary
 To quantify percentage of edited alleles in peripheral blood leukocytes and bone marrow cells
 To assess the production of HbF post-CTX001 infusion
 To assess the effects of infusion of CTX001 on disease-specific events and clinical status
Exploratory
 To assess the ability of biomarkers to characterize CTX001 effect and predict treatment outcomes
Study design:
This is a single-arm, open-label, multisite, single-dose Phase 1/2 study that will enroll subjects 12 to 35 years of
age with TDT. The first part of the study will include up to 12 subjects dosed. After or more subjects have been
dosed with CTX001, the study may be expanded to dose up to a total of 45 subjects.
Stage 1: Screening and pre-mobilization period
During this period, subjects who meet the inclusion/exclusion criteria have the option to undergo fertility
preservation via cryopreservation of oocyte or sperm, or gonadal tissue banking, if appropriate per subject age
and local site practice.
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Stage 2: Mobilization, autologous CD34+ stem cell collection, CTX001 manufacture and disposition
After eligibility has been confirmed, each subject will undergo stem cell mobilization with a combination of
granulocyte-colony stimulating factor (G-CSF; e.g. filgrastim) and plerixafor. Peripheral blood mononuclear cells
(PBMCs) will be collected by apheresis. Subjects will undergo apheresis for 3 consecutive days to collect CD34 +
hHSPC. The targeted CD34+ cell collection is at least 15 x 106 CD34+ cells/kg for manufacturing of CTX001 in
order to achieve a minimum target dose of 3 × 10 6 CD34+ cells/kg. An additional 2 x 106 CD34+ cells/kg will be
collected as backup for rescue therapy in an event of non-engraftment with CTX001. All collected cells intended
for manufacturing will be shipped daily at 2-8oC to the manufacturing facility. Cells collected for rescue therapy
will not undergo the editing manufacturing process and will be cryopreserved at the site.
If sufficient numbers of cells for CTX001 manufacturing and backup are not obtained (as determined based on
discussion between the Sponsor and investigator), up to 2 additional mobilization and apheresis cycles will be
allowed to collect additional cells. The additional mobilization and apheresis cycle may be performed at least 14
days after the first day of the prior mobilization cycle and no more than 60 days after the end of the prior cycle.
Stage 3: Myeloablative conditioning (Stage 3A) and infusion of CTX001 (Day 1, Stage 3B)
Stage 3A - Myeloablative conditioning: After the CTX001 product is received at the site and it has been
confirmed that the backup cells remain available and in suitable condition to be administered if needed, the
subject will be hospitalized to undergo myeloablative conditioning with busulfan.
Busulfan will be administered intravenously (IV) daily at a starting dose of 3.2 mg/kg/day for 4 consecutive days.
Once-daily dosing is the preferred schedule; however, the busulfan dosing regimen may be adjusted to be given
as 0.8 mg/kg every 6 hours (Q6H) for 4 consecutive days per the site’s standard practice.
The dose of busulfan may be adjusted based on the pharmacokinetics (PK) of the first busulfan dose to maintain
appropriate levels for myeloablation. The average target area under the curve (AUC) for subjects receiving a
starting dose of 3.2 mg/kg/day for 4 days is 5000 µM*min (range: 4500 to 5500 µM*min); equivalent to target
cumulative busulfan exposure of 90 mg x hr/L (range 80-100 mg x hr/L). The mean AUC for subjects
administered busulfan Q6H for 4 days is 1125 µM*min (range: 900 to 1350 µM*min). For subjects less than
34 kg, institutional and/or regional practice for busulfan dosing may be used.
Clinical sites may use a test dose of busulfan within 30 days (consistent with institutional guidelines) prior to
initiation of busulfan to pre-determine busulfan dose.
Stage 3B - CTX001 infusion:
Infusion of CTX001 will occur at a minimum of 48 hours following the completion of busulfan infusion and at a
maximum of 7 days after the completion of busulfan infusion.
On Day 1, each vial will be administered no more than 20 minutes from time of thaw. The entire dose of
CTX001, which at a minimum will be 3.0 × 106 CD34+ cells/kg, will be infused through a central venous
catheter. All vial(s) containing CTX001 should be infused.
Stage 4: Follow-up, through engraftment and up to 24 months after CTX001 product infusion
Stage 4A - Post-infusion until hospital discharge (post-engraftment): Subjects will be followed daily in the
transplant unit and receive supportive care according to standard practices for subjects undergoing hematopoietic
stem cell transplant (HSCT). Subjects will be monitored for adverse events (AEs) and engraftment. Packed RBC
and platelet transfusions will be given to subjects per standard practices/investigator judgement for subjects
undergoing HSCT. Subjects will be discharged from the transplant unit upon neutrophil engraftment (defined as
absolute neutrophil count [ANC] ≥500/µL for 3 measurements on 3 consecutive days) and stabilization of major
medical issues as per local hospital guidelines and/or investigator judgment.
Stage 4B - Post-engraftment to 2-years post-infusion: Stage 4B starts after subjects have successfully engrafted,
are clinically stable, and have been discharged from the transplant unit. Subjects will be followed for 24 months
after CTX001 infusion with physical exams, laboratory and imaging assessments, and AE evaluations. Subjects
will be allowed to restart iron chelation approximately 1 month after CTX001 infusion according to the site’s
management guidelines and/or investigator judgment.
Following engraftment, transfusions of packed RBCs should be avoided for hemoglobin (Hb) ≥9 g/dL, unless
medically indicated (e.g., symptomatic anemia or as a requirement for surgery). It is recommended that subjects
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should receive packed RBC transfusions for Hb <7.0 g/dL, while medical judgement is advised to transfuse for
Hb levels of 7-9 g/dL based on a subject’s clinical needs.
Following receipt of busulfan conditioning and CTX001, subjects should undergo at least yearly PEs, and receive
screening for malignancy based on appropriate country-specific cancer guidelines and subject medical
history. Subjects should also undergo appropriate malignancy evaluation if they have unexplained symptoms,
signs, or laboratory abnormalities that could be related to an underlying malignancy. Examples include
unexplained weight loss or fever, lymphadenopathy, and abnormal blood counts. The medical monitor should be
notified if a malignancy is found.
All subjects infused with CTX001 will be asked to enroll in a long-term follow-up study
(Study VX18-CTX001-131) following completion or withdrawal/discontinuation.
Data Monitoring Committee: A data monitoring committee (DMC) will be formed before the first subject is
dosed. The DMC’s objectives and operational details will be defined in a separate document (DMC charter).
The DMC will be charged with ensuring the safety of the subjects, safeguarding their interests, and ensuring the
quality and integrity of the trial. The DMC will conduct reviews of study data as outlined in the DMC charter.
The DMC may recommend that the Sponsor suspend enrollment, amend the study, or discontinue the study at
any time.
The DMC will review available engraftment and safety data for the first subject before the second subject will
undergo myeloablation. If the second subject infused with CTX001 experiences Grade ≥3 AEs other than those
typically associated with busulfan conditioning or autologous transplant procedure through engraftment or
30 days post infusion, whichever is longer, the DMC will also review data from the second subject before the
remaining subjects can undergo myeloablation and CTX001 infusion.
In addition, in order to recommend whether to expand the study to subjects 12 to <18 years of age, the DMC
will review of efficacy and safety data following CTX001 infusion from at least subjects
≥18 years old.
Further, the DMC will review safety and efficacy data after at least subjects have received CTX001 infusion
in order to recommend whether or not to expand the study to dose up to 45 subjects.
The Sponsor or designee will be responsible for promptly alerting the DMC regarding any suspected, unexpected,
serious adverse reaction (SUSAR).
Number of Subjects and Enrollment Plan: In the first part of the study, up to 12 adult TDT subjects will be
enrolled and treated with the CTX001.
The first 2 subjects will be non-β0/β0 and will be treated with CTX001 in a staggered manner to ensure that
there is successful engraftment of the first subject before treating the second subject in the study. The second
subject will not undergo myeloablation until the first subject achieves neutrophil engraftment and the available
engraftment and safety data have been reviewed by the DMC and at least 30 days after infusion of CTX001 to
the first subject. If the second subject infused with CTX001 has not experienced Grade ≥3 AEs other than those
typically associated with busulfan conditioning or autologous transplant procedure through engraftment or
30 days post infusion (whichever is longer), the remaining subjects, which may include subjects with the β0/β0
genotype, can undergo conditioning and CTX001 infusion concurrently. In the event that the second subject
infused with CTX001 experiences Grade ≥3 AEs other than those typically associated with busulfan
conditioning or autologous transplant procedure, a DMC meeting will be convened, data will be reviewed, and a
substantial amendment will be submitted to the clinical trial application (CTA) before the remaining subjects
can undergo conditioning and CTX001 infusion. All steps that precede busulfan myeloablation such as consent,
screening, stem cell collection, and manufacturing may proceed concurrently without staggering subjects.
After or more subjects ≥18 of age have been dosed with CTX001 and have of efficacy and
safety data following infusion of CTX001, the study may be expanded to subjects 12 to <18 years old.
Approximately adult subjects will be dosed with CTX001 before the conditioning and dosing of the first
pediatric subject.
After or more subjects have been treated with CTX001, the trial may be expanded up to 45 subjects dosed.
All subjects infused with CTX001 will be asked to enroll into an additional long-term follow-up study
(Study VX18-CTX001-131).
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Inclusion Criteria:
1. Subjects 12 to 35 years of age, inclusive on the date of informed consent
2. Subject (or their legally authorized representative or guardian) will sign and date an informed consent form
(ICF) and, where applicable, an assent form.
a. Documented homozygous β-thalassemia or compound heterozygous β-thalassemia including β-
thalassemia/hemoglobin E (HbE). Subjects can be enrolled based on historical data, but a confirmation of the
genotype using the study central laboratory will be required before busulfan conditioning. The β 0 and non β0
genotypes are defined using the HbVar Database.
b. A history of at least 100 mL/kg/year or 10 units/year of packed RBC transfusions in the prior 2 years
before signing the consent or the last rescreening for patients going through repeat screening.
4. Karnofsky performance status of ≥80% for subjects ≥16 years of age. Lansky performance status of ≥80%
for subjects <16 years of age.
6. Access to detailed medical records on packed RBC transfusions, including volume or units of packed RBCs
and associated pre-transfusion Hb values, and in-patient hospitalizations, for at least the 2 years prior to consent.
7. Female subjects of childbearing potential (postmenarcheal, has an intact uterus and at least 1 ovary, and is
less than 1 year postmenopausal) must agree to use acceptable method(s) of contraception from consent through
at least 6 months after CTX001 infusion.
8. Male subjects of reproductive capacity must agree to use effective contraception from start of mobilization
through at least 6 months after CTX001 infusion (Section 3.6).
9. Willing and able to comply with scheduled visits, treatment plan, laboratory tests, contraceptive guidelines,
and other study procedures.
10. Willing to participate in an additional long-term follow-up study (Study VX18-CTX001-131) after
completion of this study.
Exclusion Criteria:
2. Prior allogeneic HSCT.
3. Subjects with associated α-thalassemia and >1 alpha chain deletion or alpha multiplications.
4. Subjects with sickle cell β-thalassemia variant.
5. Clinically significant and active bacterial, viral, fungal, or parasitic infection as determined by the
investigator.
6. White blood cell count <3 × 109/L or platelet count <50 × 109/L not related to hypersplenism per investigator
judgment.
8. History of any illness or any clinical condition that, in the opinion of the investigator, might confound the
results of the study or pose an additional risk in administering study drug to the subject. This may include,
but is not limited to: immediate family member with a known family cancer syndrome, history of relevant
drug allergies; history of cardiovascular or central nervous system disease; history or presence of clinically
significant pathology; history of uncontrolled seizure disorders, or history of psychiatric disorders.
9. Any prior or current malignancy or myeloproliferative disorder or a significant immunodeficiency disorder.
a. Aspartate transaminase (AST), alanine transaminase (ALT) >3 x the upper limit of normal (ULN), or
direct bilirubin value >2.5 x the ULN, or:
c. History of cirrhosis or any evidence of bridging fibrosis, or active hepatitis on previous liver biopsy.
d. Liver iron content (LIC) ≥15 mg/g on R2* MRI of liver.
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11. A cardiac T2* <10 ms by MRI or left ventricular ejection fraction (LVEF) <45% by echocardiogram.
13. Diffusion capacity of the lungs for carbon monoxide (DLco) <50% of predicted (corrected for hemoglobin
and/or alveolar volume).
14. Prior treatment with gene therapy/editing product.
15. Intolerance, contraindication, or known sensitivity to plerixafor, G-CSF products (e.g., filgrastim), or
busulfan. Prior anaphylaxis with excipients of CTX001 product (dimethyl sulfoxide [DMSO], Dextran).
16. Positive for the presence of human immunodeficiency virus-1 (HIV-1) or human immunodeficiency virus-2
(HIV-2) (positive antigen/antibody AND nucleic acid tests [NAT]), hepatitis B virus (HBV) (positive
hepatitis B core antibody [HBcAb] AND NAT tests), syphilis [positive screening AND confirmatory tests],
or hepatitis C virus (HCV positive antibody [HCAb] and NAT tests). Additional infectious disease markers
should be obtained and tested as required by the local authority for the collection and processing of cellular
therapy products. These additional tests (e.g., HTLV-1, HTLV-2, malaria, tuberculosis, toxoplasmosis,
Trypanosoma cruzi, or West Nile virus) will be evaluated to determine overall impact to the patient and
manufacturing of CTX001.
17. Participation in another clinical study with an investigational drug/product within 30 days of screening or
fewer than 5 half-lives of the investigational agent, whichever is longer from screening.
18. An assessment by the investigator that the subject would not comply with the study procedures outlined in
the protocol.
Duration of Subject Participation: Duration of Stage 1 will be approximately 1 to 3 months; Stage 2

approximately 2 to 3 months; Stage 3 approximately 1 month; Stage 4 approximately 2 years. Subjects will be
followed on study for a total of approximately 2 years after CTX001 infusion.
All subjects will be asked to enroll in a long-term follow-up study (Study VX18-CTX001-131) following
completion or withdrawal/discontinuation from this study.
Test Product, Dose, and Mode of Administration: CTX001 product is composed of autologous CD34 +
hHSPCs modified with CRISPR-Cas9 at the erythroid lineage-specific enhancer of the BCL11A gene. CTX001
will be formulated in CryoStor CS5 medium which contains 5% DMSO and Dextran 40. Subjects will receive the
entire dose of CTX001, which will be at a minimum of 3.0 × 106 CD34+ cells/kg, through a central venous
catheter.
Study Endpoints
Safety Endpoints:
 Successful neutrophil engraftment within 42 days after CTX001 infusion.
 Time to neutrophil engraftment.
 Time to platelet engraftment
 Safety and tolerability assessments based on adverse events (AEs), clinical laboratory values, and vital signs
 Incidence of transplant related mortality (TRM) within 100 days and within 1 year post-CTX001 infusion.
 All-cause mortality.
Primary Efficacy Endpoint
 Proportion of subjects achieving sustained transfusion reduction for at least 6 months (sustained TR6) at the
time of analysis starting 3 months after CTX001 infusion.
Key Secondary Efficacy Endpoint

 Proportion of subjects achieving sustained transfusion independence for at least 6 months (sustained TI6) at
the time of analysis starting 3 months after CTX001 infusion.
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TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES

TABLE OF CONTENTS
Protocol Approval Signature Page .............................................................................................. 2
Protocol Approval Signature Page .............................................................................................. 3
Protocol Acceptance Form ........................................................................................................... 4
Summary of Changes to the Protocol......................................................................................... 5
Synopsis.......................................................................................................................................... 8
Key Secondary Efficacy Endpoint ........................................................................................ 12
Secondary Endpoints ............................................................................................................ 13
Exploratory Endpoints .......................................................................................................... 13
Table of Contents, List of Tables, and List of Figures Table of Contents ............................. 14
List of Abbreviations and Definitions of Terms ....................................................................... 18
1. Introduction.......................................................................................................................... 22
1.1. β-thalassemia .................................................................................................................. 22
1.2. Current Treatment Approaches for Transfusion-Dependent β-Thalassemia (TDT) ...... 24
1.3. CRISPR-Cas9 Gene Editing Approach .......................................................................... 25
1.4. BCL11A ......................................................................................................................... 27
1.5. Investigational Product: CTX001 ................................................................................... 27
1.6. Non-clinical Experience ................................................................................................. 28
1.7. Clinical Experience ........................................................................................................ 29
1.8. Therapeutic Rationale ..................................................................................................... 29
1.9. Dose Rationale................................................................................................................ 31
1.10. Risk Assessment ............................................................................................................. 31
1.10.1. Potential Risks of CTX001 ..................................................................................... 31
1.10.2. Potential Risks of Mobilization, Apheresis and Autologous Transplantation........ 32
2. Study Objectives and Endpoints ........................................................................................ 33
2.1. Objectives ....................................................................................................................... 33
2.1.1. Primary ................................................................................................................... 33
2.1.2. Secondary ............................................................................................................... 34
2.1.3. Exploratory ............................................................................................................. 34
2.2. Endpoints ........................................................................................................................ 34
2.2.1. Safety Endpoints ..................................................................................................... 34
2.2.2. Primary Efficacy Endpoint ..................................................................................... 34
2.2.3. Key Secondary Efficacy Endpoint ......................................................................... 34
2.2.4. Secondary Endpoints .............................................................................................. 34
2.2.5. Exploratory Endpoints ............................................................................................ 35
3. Study Population.................................................................................................................. 35
3.1. Number of Subjects ........................................................................................................ 35
3.2. Subject Inclusion Criteria ............................................................................................... 36
3.3. Subject Exclusion Criteria .............................................................................................. 36
3.4. Enrolled Subjects and Screen Failures ........................................................................... 38
3.5. Waivers of Inclusion/Exclusion Criteria ........................................................................ 38
3.6. Contraception ................................................................................................................. 38
3.7. Subject Identification...................................................................................................... 41
3.8. Subject Withdrawal ........................................................................................................ 41
3.9. Replacement of Subjects ................................................................................................ 42
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4. Study Design ......................................................................................................................... 42

4.1. Study Overview .............................................................................................................. 42
4.2. Study Duration................................................................................................................ 45
4.3. Study Enrollment Plan .................................................................................................... 45
5. Study Stopping Rules .......................................................................................................... 46
5.1. Enrollment Suspension Criteria ...................................................................................... 46
5.2. Stopping Rules................................................................................................................ 47
5.3. End of Study Definition.................................................................................................. 47
5.4. Sponsor’s Termination of Study ..................................................................................... 48
6. Treatment Plan .................................................................................................................... 48
6.1. Description of CTX001 .................................................................................................. 48
6.2. Mobilization and Apheresis ............................................................................................ 48
6.2.1. Mobilization............................................................................................................ 48
6.2.2. Apheresis Procedure ............................................................................................... 50
6.3. Shipment to Manufacturing Facility ............................................................................... 50
6.4. Manufacture of CTX001 ................................................................................................ 50
6.5. Shipment of CTX001 Product to Treatment Site ........................................................... 51
6.6. Storage of CTX001 Product ........................................................................................... 51
6.7. Conditioning: Busulfan Administration ......................................................................... 51
6.8. CTX001 Infusion Procedures, Dose, and Administration .............................................. 52
6.9. Post-CTX001 Infusion Infection Prophylaxis and Surveillance .................................... 53
6.10. Screening for Malignancy .............................................................................................. 53
6.11. Prior and Concomitant Treatments and Procedures ....................................................... 53
6.11.1. Prior medications .................................................................................................... 53
6.11.2. Venous Access ........................................................................................................ 53
6.11.3. Transfusions............................................................................................................ 53
6.11.4. Iron Chelation ......................................................................................................... 54
6.11.5. Prohibited medications ........................................................................................... 54
6.12. CTX001 Accountability ................................................................................................. 54
6.13. Treatment Compliance ................................................................................................... 55
6.14. Allelic Editing of CTX001 Drug Product ...................................................................... 55
7. Visit evaluation schedule ..................................................................................................... 56
8. Study Assessments ............................................................................................................... 62
8.1. Demographic/Medical History ....................................................................................... 62
8.2. Transfusions ................................................................................................................... 62
8.3. Vital Signs and Weight/Height ....................................................................................... 62
8.4. Physical Examination ..................................................................................................... 62
8.5. Electrocardiogram .......................................................................................................... 62
8.6. Diffusing Capacity of the Lungs for Carbon Monoxide (DLCO) .................................. 63
8.7. Imaging Studies .............................................................................................................. 63
8.8. Adverse Events ............................................................................................................... 63
8.9. Laboratory Assessments ................................................................................................. 63
8.10. Blood for Biomarker Assessments ................................................................................. 65
8.11. Blood for Exploratory Research Studies and Assay Development ................................ 65
8.12. Sperm and Oocyte Banking ............................................................................................ 66
8.13. Biopsies .......................................................................................................................... 66
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8.13.1. Liver Biopsy ........................................................................................................... 66

8.13.2. Bone Marrow Aspirate ........................................................................................... 66
8.14. Patient Reported Outcomes (PROs) ............................................................................... 66
8.14.1. EQ-5D-5L and EQ-5D-Y ....................................................................................... 66
8.14.2. FACT-BMT ............................................................................................................ 67
8.14.3. Pediatric Quality of Life Inventory (PedsQL) ........................................................ 67
8.15. Mini-Mental State Examination and Wechsler Abbreviated Scale of Intelligence ........ 68
9. Safety Monitoring and reporting ....................................................................................... 68
9.1. Adverse and Serious Adverse Events ............................................................................. 68
9.1.1. Definition of Adverse Events ................................................................................. 68
9.1.2. Reporting Serious Adverse Events ......................................................................... 72
9.1.3. Expedited Reporting and Investigator Safety Letters ............................................. 73
9.2. Pregnancy ....................................................................................................................... 73
9.3. Data Monitoring Committee........................................................................................... 74
10. Statistical and Analytical Plans .......................................................................................... 74
10.1. Sample Size Estimation .................................................................................................. 74
10.2. Analysis Sets .................................................................................................................. 74
10.3. General Considerations .................................................................................................. 75
10.4. Subject Disposition ......................................................................................................... 75
10.5. Demographic and Baseline Characteristics .................................................................... 76
10.6. Efficacy Analysis............................................................................................................ 76
10.6.1. Analysis of Primary Efficacy Endpoint .................................................................. 76
10.6.2. Analysis of Key Secondary Endpoint ..................................................................... 76
10.6.3. Secondary Efficacy Endpoints................................................................................ 76
10.7. Safety Analysis ............................................................................................................... 78
10.7.1. Engraftment ............................................................................................................ 78
10.7.2. Adverse Events ....................................................................................................... 78
10.7.3. Clinical Laboratory Assessments ........................................................................... 79
10.7.4. Electrocardiogram .................................................................................................. 79
10.7.5. Vital Signs .............................................................................................................. 79
10.7.6. Physical Examination ............................................................................................. 79
10.8. Exploratory Analysis ...................................................................................................... 79
10.9. Interim Analyses ............................................................................................................. 80
10.10. Procedures for Handling Missing Data .................................................................. 80
10.11. Steering Committee ................................................................................................ 81
10.12. Endpoint Adjudication Committee (EAC) ............................................................. 81
11. Data collection and management ....................................................................................... 81
11.1. Monitoring ...................................................................................................................... 81
11.2. Electronic Data Capture (EDC) ...................................................................................... 82
11.3. Audits and Inspections ................................................................................................... 82
12. QUALITY CONTROL AND QUALITY ASSURANCE ................................................. 83
13. Ethical And Regulatory Considerations ............................................................................ 83
13.1. Ethics Committee (EC) / Institutional Review Board (IRB) .......................................... 83
13.2. Ethics Review ................................................................................................................. 83
13.3. Conduct of the Study ...................................................................................................... 83
13.4. Written Informed Consent .............................................................................................. 83
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LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

The following abbreviations and specialist terms are used in this study protocol.
Table 1: Abbreviations and Specialist Terms
Ab Antibody
ADL Activities of daily living
AE Adverse event
Allo-HSCT Allogeneic hematopoietic stem cell transplant
ALT Alanine transaminase
ANC Absolute neutrophil count
aPTT Activated partial thromboplastin time
AST Aspartate transaminase
AUC Area under the curve
BM Bone Marrow
bpm Beats per minute
CBC Complete blood count
cGMP Current Good Manufacturing Practice
CIC Cardiac iron content
CRF Case report form
CRISPR-Cas9 CRISPR-Cas9 means: Clustered Regularly Interspaced Short
Palindromic Repeats-CRISPR associated 9 nuclease
crRNA crisprRNA
CTX001 Autologous CRISPR-Cas9 modified CD34+ hHSPCs
DLco Diffusing capacity of the lungs for carbon monoxide
DMC Data Monitoring Committee
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DSB Double-strand break
EC Ethics Committee
ECG Electrocardiogram
eCRF Electronic case report form
EMA European Medicines Agency
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EQ-5D-5L EuroQol Questionnaire – 5 dimensions – 5 levels of severity
EQ-5D-Y EuroQol Questionnaire – 5 dimensions – youth
FACT-BMT Functional assessment of cancer therapy-bone marrow
transplant
FSH Follicle-stimulating hormone
FTA-ABS Fluorescent treponemal antibody absorption test
G-CSF Granulocyte-colony stimulating factor
GGTP Gamma-glutamyl transpeptidase
GPS (Vertex) Global Patient Safety
Genome-wide Unbiased Identification of DSB Enabled by
GUIDE-seq
sequencing
GvHD Graft-versus-host disease
Hb Hemoglobin
HbA Hemoglobin A or adult hemoglobin
HBB Human B globin
HBcAb Hepatitis B core antibody
HbE Hemoglobin E
HbF Fetal hemoglobin
HBV Hepatitis B virus
HCAb Hepatitis C virus antibody
HCV Hepatitis C virus
HDR Homology-directed repair
hHSPCs Human hematopoietic stem and progenitor cells
HLA Human leukocyte antigen
HSCT Hematopoietic stem cell transplant
HTLV-1 Human T cell leukemia virus-1
HTLV-2 Human T cell leukemia virus-2
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IA Interim Analysis
ICF Informed Consent Form
ICH International Conference on Harmonisation
INR International normalized ratio
IRB Institutional Review Board
IV Intravenous / intravenously
kg Kilogram
LDH Lactate dehydrogenase
LIC Liver iron concentration
LT-HSC Long-term hematopoietic stem cells
LVEF Left ventricular ejection fraction
μg Microgram
M Month
Max Maximum
MCH Mean corpuscular hemoglobin
MCHC Mean corpuscular hemoglobin concentration
MCV Mean corpuscular volume
Min Minimum
mL Milliliter
MRI Magnetic resonance imaging
mRNA Messenger ribonucleic acid
N Number of subjects
NAT Nuclei acid testing
NCI National Cancer Institute

NHEJ Non-Homologous End-Joining
NSG NOD SCID gamma
nt Nucleotide
NTDT Non-transfusion-dependent thalassemia
PAM Protospacer adjacent motif
PBMC Peripheral blood mononuclear cell
PFT Pulmonary function test
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PedsQL Pediatric Quality of Life
pH Hydrogen ion concentration
PI Principal Investigator
PK Pharmacokinetic(s)
PRO Patient reported outcome
PT Prothrombin time
PTT Partial thromboplastin time
Q6H Every 6 hours
RBC Red blood cell
RDW Red blood cell distribution width
RNA Ribonucleic acid
RPR Rapid plasma reagin
SAE Serious adverse event
SAP Statistical Analysis Plan
SD Standard Deviation
sgRNA Single-guide RNA
SOP Standard Operating Procedure
SUSAR Suspected unexpected serious adverse reaction
TDT Transfusion-dependent β-thalassemia
TE Treatment emergent
TI Transfusion independence
TIBC Total iron binding capacity
TR Transfusion reduction
TRM Transplant-related mortality
ULN Upper limit of normal
VAS Visual analog scale
VOD Veno-occlusive disease
WBC White blood cell
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1. INTRODUCTION
CTX001 is a product consisting of autologous CD34+ human hematopoietic stem and progenitor
cells (hHSPCs) modified by Clustered Regularly Interspaced Short Palindromic Repeats-
CRISPR associated 9 nuclease (CRISPR-Cas9)-mediated gene editing at the erythroid lineage-
specific enhancer region of the B-cell Lymphoma/Leukemia 11A (BCL11A) gene located on
intron 2, between exons 2 and 3, on chromosome 2. The disruption of the BCL11A regulatory
sequences allows for reactivation of transcription and protein expression of γ-globin, resulting in
increased levels of fetal hemoglobin (HbF). In severe β-thalassemia, increased HbF decreases the
total hemoglobin (Hb) deficit in red blood cells (RBCs) and increased γ-globin production
improves the α-globin to non-α-globin ratio imbalance, thereby reducing ineffective
erythropoiesis (due to reduced uncomplexed α-globin chains), and prolonging erythrocyte
survival (due to decrease in hemolysis). This results in improvement of anemia and reduction in
transfusion needs in β-thalassemia patients, similar to a naturally occurring state of Hereditary
Persistence of Fetal Hemoglobin (HPFH).
1.1. β-thalassemia
β-thalassemia is one of the most common autosomal recessive disorders worldwide with high
prevalence in populations in the Mediterranean (5-15%), Middle-East and West Asia (2-5%),
South-East Asia (up to 10%), and South Asia (up to 18%) (Colah et al., 2010). Due to population
migration, β-thalassemia is also found in Northern Europe, North and South America, Caribbean,
and Australia. Currently, the worldwide living population of β-thalassemia major patients is
estimated to be 200,000 that are registered and receiving treatment (Galanello and Origa, 2010).
β-thalassemia is caused by a spectrum of mutations that result in reduced or absent production of
adult hemoglobin (HbA). Different forms of Hb are produced during different stages of
development. Fetal hemoglobin (HbF) is the predominant Hb prior to birth and extending into
the newborn period. HbF is a tetrameric globin protein containing 2 γ-globin and 2 α-globin
chains (α2γ2). After the newborn period, the main form of Hb is HbA (Figure 1), a
heterotetramer comprised of 2 β-globin and 2 α-globin chains (α2β2). HbA normally accounts
for > 95% of the total Hb in the blood of adults.
Figure 1 Molecular Structure of Hemoglobin A (OpenStax College, 2016)

The degree of impaired HbA production, resulting from the extent of incomplete (β+) or absent
(β0) β-globin expression, determines the severity of β-thalassemia.
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erythropoiesis due to increased HbF levels may also have a positive effect on iron overload and
end-organ damage (Tanno and Miller, 2010).
1.2. Current Treatment Approaches for Transfusion-Dependent

β-Thalassemia (TDT)
Treatment of TDT includes lifelong blood transfusions every 3-6 weeks. The aim of transfusion
therapy is to keep Hb levels ≥9 g/dL in order to ameliorate the symptoms and physiologic
sequalae of severe anemia and to maintain normal growth and development (Rachmilewitz and
Giardina et al, 2011). Though chronic blood transfusion regimens are effective at preventing the
hallmark symptoms and physical manifestations of disease, they introduce a large iron overload
(Cao et al., 1996) that may lead to mortality through iron associated heart and liver toxicity
(Vichinsky et al., 2005) To prevent this, iron overload must be managed with iron chelation
regimens that are usually initiated at an early age (Saliba et al., 2015). Poor compliance with
chelation regimens remains a key challenge. Despite the improvements with current therapies,
there is poor quality of life and overall survival until the age of 30 years is only 55% (Modell et
al., 2000; Delea et al., 2007).
Currently, the only curative treatment options for TDT are allogeneic hematopoietic stem cell
transplant (allo-HSCT) and a recently approved lentiviral gene therapy
(betibeglogene autotemcel; Zynteglo®). There are significant risks associated with allo-HSCT
such as serious infections, graft failure and graft-versus-host disease (GvHD), some of which can
be fatal. As such, transplants are infrequently performed, and are offered primarily to subjects
who have available human leukocyte antigen (HLA)-matched sibling donors, who are young
(<16 years of age), and who do not have significant iron overload. Because of the need of an
HLA-matched sibling donor, allo-HSCT is available to only <25% of eligible patients with
remainder of the patients requiring lifelong transfusions and chelation. Transplants using
alternative donor sources such as unrelated cord blood and haploidentical donors remain
experimental due to higher risk of engraftment failure and GvHD (Mathews et al., 2014).
The absence of suitable donors, the significant risks associated with transplantation, and the
requirement for post-transplant immunosuppression therapy to prevent GvHD indicate an unmet
medical need for novel therapies with transformative potential for subjects with TDT.
Gene-based therapies are promising approaches that have the potential to provide a functional
cure in patients with severe β-thalassemia. There is currently 1 approved lentivirus-based gene
therapy for the treatment of TDT (betibeglogene autotemcel), which was approved in 2019. The
integrating lentiglobin vector relies on random insertion into the genome for production of a
mutated form of hemoglobin (HbA T87Q) (Zynteglo Summary of Product Characteristics).
However, this therapy is not a treatment option for all patients with TDT, and studies are still in
progress to evaluate the long-term efficacy of this newly approved therapy. Additional curative
therapies are needed.
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1.3. CRISPR-Cas9 Gene Editing Approach

been repurposed as a ribonucleic acid (RNA)-guided deoxyribonucleic acid (DNA)-targeting
platform used for gene editing (Jinek et al., 2012).
The CRISPR-Cas9 system from Streptococcus pyogenes relies on only one protein, the nuclease
Cas9, and two noncoding RNAs, crisprRNA (crRNA) and tracrRNA, to target DNA.
 crRNA drives target sequence recognition and specificity of the CRISPR-Cas9 complex
through Watson-Crick base pairing with a (typically) 20 nucleotide (nt) sequence in the
target DNA. The 5’ 20nt of crRNA is responsible for this DNA recognition. Changing
these 20nt in the crRNA alters the sequence specificity of the CRISPR-Cas9 complex and
allows sequence-specific targeting of other loci (targets).
 TracrRNA hybridizes with the 3’ end of crRNA to form an RNA duplex structure that is
then bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9
complex which can then cleave the target DNA.
These two noncoding RNAs can further be joined by a 4nt linker loop to form a chimeric RNA
called single-guide RNA (sgRNA) (Figure 3). The CRISPR-Cas9 (sgRNA/Cas9) complex
together forms a ribonucleoprotein (RNP).
The CRISPR-Cas9 (sgRNA/Cas9) complex binds double-stranded DNA sequences that contain a
sequence match to the first 20nt of the sgRNA if the target sequence is followed by a protospacer
PAM is a short DNA motif immediately adjacent to the target DNA sequence that the Cas9
endonuclease requires for recognition of the sequence as a target site (Jinek et al., 2012).
Figure 3 Schematic of the CRISPR-Cas9 Complex

Complex containing a single gRNA wherein the crRNA and tracrRNA are joined by a linker loop (adapted from Jinek
et al., 2012).
Once CRISPR-Cas9 (sgRNA/Cas9) complex is bound to DNA at a target site, two independent
nuclease domains in Cas9 will each cleave one of the DNA strands 3 bases upstream of the PAM
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site, leaving a blunt end DNA double-strand break (DSB) (Jinek et al., 2012).
Formation of the site-specific DSB is the first of two key steps in genome editing. The second
key step is repair of the DSB. Cells use 2 main DNA repair pathways to resolve the DSB: non-
homologous end-joining (NHEJ) and homology-directed repair (HDR) (Figure 4).
including non-dividing cells. However, NHEJ is error-prone and can often result in the removal
or addition of between one and several hundred nucleotides at the site of the DSB, though such
modifications are typically <20nt (Jinek et al., 2012). If these small insertions/deletions (indels)
occur within an open-reading frame, there is a high likelihood that the gene will be functionally
disrupted due to frame shift or deletion of critical amino acid residues from the expressed
protein. Similarly, disruption of critical regulatory elements can significantly alter the expression
level of target genes.
Figure 4 CRISPR-Cas9-Mediated Genome Editing Strategies

CRISPR-Cas9-mediated DNA cleavage creates a double-strand break (DSB) at the target locus. The DSB can be
repaired by non-homologous end-joining (NHEJ) and homology-directed repair (HDR) (Genome Editing Strategy
Image, 2015).
CTX001 relies on DNA repair by NHEJ to create insertion or deletion (indels) of one or more
nucleotides at the site of the DSB within the BCL11A enhancer locus. When the critical regulatory
elements are modified, they can significantly alter the expression level of BCL11A. The intended
gene modification employed by CTX001 disrupts an essential transcription factor binding site
(GATA1), leading to reduced BCL11A expression and the downstream effect of HbF induction.
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1.4. BCL11A
BCL11A gene encodes a transcription factor, highly expressed in the brain, B-lymphocytes, and
adult erythroid precursor lineages, that acts as repressor of human -globin gene and exerts this
effect by binding directly to the globin locus on chromosome 11. Sequence variants within the
BCL11A gene (located on chromosome 2) were demonstrated to correlate with HbF expression
in genome-wide association studies (Menzel et al., 2007; Lettre et al., 2008; Uda et al., 2008).
Knockdown of BCL11A in primary human erythroid precursor cells resulted in increased HbF
(Sankaran et al., 2008). Furthermore, BCL11A knockout was shown to disrupt developmental
silencing of human fetal γ-globin in transgenic mice (Sankaran et al., 2009). Conditional
knockout in adult sickle cell disease transgenic mice leads to reactivation of γ-globin and
amelioration of symptoms (Xu et al., 2011).
Haploid missense mutations in BCL11A gene are associated with persistent HbF ranging from
8.7-20.8% (Dias et al., 2016), and haploinsufficiency in BCL11A is associated with persistent
HbF ranging from 3.1-29.7%, and where measured, approximately 50% reduction in BCL11A
messenger ribonucleic acid (mRNA) (Basak et al., 2015; Funnell et al., 2015; Dias et al., 2016)
in non-thalassemic patients.
CRISPR-Cas9-mediated disruption of the BCL11A intronic erythroid enhancer in human
erythroid precursors led to a 20% increase above background in HbF expression (Canver et al.,
2015) These data form the approach of CTX001 to pursue disruption of BCL11A erythroid
enhancer as a target for CRISPR-Cas9 modification to re-express HbF in subjects with TDT.
1.5. Investigational Product: CTX001

CTX001 is a cellular product consisting of autologous CD34+ human hematopoietic stem and
progenitor cells (hHSPCs) modified by CRISPR-Cas9-mediated gene editing. The target of the
CRISPR-Cas9 gene editing is the erythroid lineage-specific enhancer region of the BCL11A
gene located on intron 2 between exons 2 and 3 on chromosome 2. More specifically, the edits
created with the highly specific guide, SPY101, target a critical transcription factor binding site
(GATA1) at an erythroid lineage-specific enhancer region (DNase I hypersensitive site +58,
DHS+58) of the BCL11A gene. Since, SPY101-RNP precisely targets the non-coding erythroid
lineage-specific enhancer region of the BCL11A gene and not the BCL11A coding sequence
itself , SPY101-RNP is expected to modulate the levels of expression of the BCL11A gene and
protein in cells solely of the erythroid lineage and not affect non-erythroid hematopoietic
lineages.
SPY101-RNP introduces DSBs in DNA in a sequence-dependent manner. Repair of the DSB by

NHEJ results in DNA insertion/deletions (indels), intended to disrupt GATA1 binding, thereby
lowering BCL11A transcription, with concomitant increases in γ-globin and fetal hemoglobin
levels. This leads to subsequent decrease in ineffective erythropoiesis (due to a reduction in
uncomplexed α-globin chains) and improved erythrocyte survival (due to decrease in hemolysis),
and ultimately amelioration of sequelae of anemia and reduction in transfusion needs (Musallam
et al., 2012). This amelioration of clinical symptoms occurs in patients with β-thalassemia and
HPFH and forms the basis for the therapeutic approach of CTX001 (Figure 5).
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depending on the cumulative amount of RBCs administered, compliance with chelation, and use
of appropriate screening (Pippard at al., 1993; Borgna-Pignatti et al., 2004). Patients who do not
adequately receive transfusion therapy may exhibit growth and maturity delays within the first
few years of life (Vogiatzi et al., 2009; Fun et al., 2010). However, without chelation, patients
may also develop severe liver and cardiac iron overload before the age of 10, and iron-related
endocrinopathy complications also result in delayed puberty, gonadal failure, and low bone
density during adolescence (Pippard et al 1993; Bajoria et al., 2011). These complications can
develop into chronic and irreversible co-morbidities that affect the TDT patient into adulthood
for the rest of their lives. Additionally, pediatric TDT patients have experienced long term
overall and disease-free survival rates that are at least as high as adult patients after allo-HSCT
(Baronciani et al., 2016; Issaragrisil et al., 2016). Therefore, an approach to TDT relying on
hematopoietic stem cell transplantation is expected to be beneficial to adolescents and children.
1.9. Dose Rationale

Total cell dose: Autologous transplantation for various indications typically employs a minimum
of 2 to 2.5 x 106 CD34+ cells/kg to support engraftment (Haynes et al., 1995; Tricot et al., 1995;
Papadopoulos et al., 1997). The Sponsor has set a conservative minimum dose of CRISPR-Cas9
edited cells that is 20-50% higher than this threshold, at ≥ 3 x 106 CD34+ cells/kg, to ensure
engraftment in all subjects. In principle, a higher number of CD34+ enriched hHSPC infused
following myeloablation is associated with more rapid engraftment, durability, and efficacy of
the treatment (Tricot et al., 1995; Shpall et al., 1998). In other gene therapy studies, such as the
recent European Medicines Agency (EMA)-approved Strimvelis (GlaxoSmithKline), doses
approaching 20 x 106 CD34+ cells/kg, an order of magnitude larger than proposed here, were
used in clinical trials with no signs of increased toxicity. Based on current manufacturing
capabilities and projected cell yields, the Sponsor proposes to set a maximum cell dose limit of
2 x 107 CD34+ cells/kg. The Sponsor, along with the Data Monitoring Committee (DMC), will
monitor for any potential dose related toxicities.
1.10. Risk Assessment

The Sponsor has carefully considered potential risks that may be associated with this novel
therapeutic approach of genetically editing hHSPCs using the CRISPR-Cas9 technology and
reinfusing CTX001 following standard myeloablative conditioning with busulfan. The Sponsor
views the overall risk to be acceptable considering the life-shortening nature, and lack of curative
or transformative therapies available for all patients with transfusion dependent β-thalassemia
and the ensuing morbidity associated with chronic RBC transfusions.
1.10.1. Potential Risks of CTX001

CRISPR-Cas9 has the proposed advantage of cleaving DNA in a highly specific manner at the
intended cutting site, the erythroid lineage-specific enhancer region of the BCL11A gene. The
intention of selecting a highly-specific guide, such as SPY101, was to reduce the risk of
tumorigenesis, the main potential risk associated with CTX001, due to potential off-target cutting
which may result in tumor suppressor inactivation or oncogene activation. Thus, when selecting
a candidate gRNA, a comprehensive system was applied to design and test gRNAs for high on-
target editing efficiency and low numbers of off-target cleavage sites.
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To assess potential risks, a non-clinical safety program was designed in accordance with the
specific requirements and recommendations for cell-based and gene therapy medicinal products
as outlined by regulatory guidance documents, taking into account the nature of the product and
its intended use (please see full description in the Investigator’s Brochure). In the non-clinical
genotoxicity and toxicology assessment of CTX001, the on-target and potential off-target editing
was extensively and systematically evaluated using multiple methods. We established that
SPY101-RNP demonstrates a high rate (approximately 88%) of on-target editing and no
detectable off-target editing at the detection level as compared to unedited cells. Karyotyping
analysis did not identify any chromosomal translocations or other detectable abnormalities. To
further demonstrate lack of tumorigenesis potential of CTX001, we conducted biodistribution
and combined toxicity and tumorigenicity studies. There was no evidence for an increased
incidence of any abnormal finding in animals receiving CTX001 compared to animals receiving
control (unedited) cells.
Overall, the thorough safety and toxicological characterization of CTX001 supports first in
human testing in the proposed clinical protocol. Although the current data do not provide any
evidence for oncogenesis, the overall risk of the CTX001 treatment process, including the
potential risk of oncogenesis, is unknown. Subjects will be informed of this potential risk and
will be monitored for up to 15 years on the long-term follow-up protocol
(Study VX18-CTX001-131).
Further details of toxicology studies performed with CTX001 pre-clinically are reported in the
accompanying Investigator’s Brochure.
1.10.2. Potential Risks of Mobilization, Apheresis and Autologous

Transplantation
Currently, the only curative treatments option for subjects with TDT is allogeneic hematopoietic
stem cell transplant (allo-HSCT) or betibeglogene autotemcel. There are significant risks
associated with allo-HSCT such as serious infections, graft failure and GvHD, some of which
can be fatal. Patients who have received betibeglogene autotemcel are not eligible for this study
with CTX001, and not all TDT patients will be candidates to receive this gene therapy.
This first-in-human study with CTX001 in subjects with TDT takes advantage of the standard
autologous transplantation platform at specialized transplant centers. Historically, autologous
stem cell transplantation is associated with 1-5% risk of mortality, which is significantly lower
than the risk of mortality associated with allo-HSCT. Sites and investigators who will participate
in this trial are well-experienced in the methods of mobilization, apheresis and infusion of
cryopreserved cell products and managing of potential adverse events (AEs) associated with
these procedures and busulfan myeloablation. This level of expertise will ensure that the
potential risks to the subjects associated with these procedures will be minimized.
Complete information regarding medications listed below is contained in the prescribing
information and should be used as a reference by the investigator to inform potential subjects and
guide monitoring and risk management.
• Risk of Filgrastim
Subjects will undergo dual mobilization with filgrastim and plerixafor. Filgrastim will be
administered intravenously or subcutaneously. Common AEs include headache, fever, muscle,
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joint and bone pains, thrombocytopenia, dyspnea, and nausea/vomiting. Enlargement or rupture
of the spleen, loss of appetite, chest pain, and low blood pressure have been reported in <10%
patients. Rare (<1%) but serious events include thrombosis and lung toxicity.
• Risk of Plerixafor
Plerixafor will be administered subcutaneously. Common AEs include injection site reaction and
bruising, diarrhea, nausea, headache, dizziness, insomnia, fatigue, and muscle, bone and joint
pain. Uncommon side effects seen in less than 1% of patients are splenic rupture, anaphylactic
reactions, and vasovagal reactions.
During mobilization subjects will be closely monitored and frequent abdominal exams will be
performed by experienced investigators and staff.
• Risk of Apheresis
All subjects will undergo apheresis following 4 days of mobilization. Apheresis may occur for
up to 3 consecutive days in order to collect the required amount of CD34+ stem cells for CTX001
manufacturing (minimum of 15 x 106 CD34+ cells/kg) and for a backup stem cell dose
(minimum of 2 x 106 CD34+ cells/kg). Apheresis will be performed per the site’s standard
procedures. Side effects associated with apheresis are related to intravascular volume changes
and electrolyte abnormalities. Most common mild to moderate side effects include paresthesias,
lethargy, dizziness, sweating, nausea and/or vomiting, arrhythmia, hypotension, and
hypocalcemia. Severe AEs may include loss of consciousness and seizures. The trial sites are
experienced in preventing and managing these potential risks.
• Risk of Busulfan Conditioning
Known risks of busulfan conditioning include infertility, myelosuppression, infections, hepatic
veno-occlusive disease (VOD), pulmonary toxicity, seizures, alopecia, and a long-term
malignancy potential. Subjects participating in the trial will be fully informed of these risks and
offered the option of cryopreservation of oocytes for females and sperm or testicular tissue for
males to facilitate management of infertility should it occur.
 Risk for pediatric subjects
Overall, the study procedures and potential risks of HSCT with CTX001 are expected to be
similar in adult and pediatric populations.
2. STUDY OBJECTIVES AND ENDPOINTS
2.1. Objectives
2.1.1. Primary
 To evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
modified CD34+ hHSPCs (CTX001) in subjects with TDT
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2.1.2. Secondary
 To quantify percentage of edited alleles in peripheral blood leukocytes and bone
marrow cells
 To assess the production of HbF post-CTX001 infusion
 To assess the effects of infusion of CTX001 on disease-specific events and clinical
status
2.1.3. Exploratory
 To assess the ability of biomarkers to characterize CTX001 effect and predict
treatment outcomes
2.2. Endpoints
2.2.1. Safety Endpoints
 Successful neutrophil engraftment within 42 days after CTX001 infusion.
 Time to neutrophil engraftment.
 Safety and tolerability assessments based on adverse events (AEs), clinical laboratory
values, and vital signs
 Incidence of transplant related mortality (TRM) within 100 days and within 1 year
post-CTX001 infusion.
 All-cause mortality.
2.2.2. Primary Efficacy Endpoint

 Proportion of subjects achieving sustained transfusion reduction for at least 6 months
(sustained TR6) at the time of analysis starting 3 months after CTX001 infusion.
2.2.3. Key Secondary Efficacy Endpoint

 Proportion of subjects achieving sustained transfusion independence for at least 6
months (sustained TI6) at the time of analysis starting 3 months after CTX001
infusion.
2.2.4. Secondary Endpoints

 Proportion of subjects achieving sustained transfusion reduction for at least 12
months (sustained TR12) at the time of analysis starting 3 months after CTX001
infusion.
 Proportion of subjects achieving sustained transfusion independence for at least
12 months (sustained TI12) at the time of analysis starting 3 months after CTX001
infusion.
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 Proportion of alleles with intended genetic modification present in peripheral blood

leukocytes over time. Intended genetic modifications are indels that modify the
sequence of the erythrocyte-specific enhancer in intron 2 of BCL11A.
 Proportion of alleles with intended genetic modification present in bone marrow cells
over time.
 Fetal hemoglobin concentration (pre-transfusion) over time.
 Total hemoglobin concentration (pre-transfusion) over time
 Change in patient reported outcomes (PROs) over time using EuroQol Quality of Life
Scale (EQ-5D-5L) for subjects ≥18 years old, EQ-5D-Y for subjects <18 years old),
functional assessment of cancer therapy-bone marrow transplant (FACT-BMT) for
subjects ≥18 years old, and Pediatric Quality of Life Inventory (PedsQL) for subjects
<18 years old.
 Change in parameters of iron overload, including:
o Liver iron concentration (LIC) from baseline as assessed by R2* magnetic
resonance imaging (MRI) and cardiac iron content (CIC) from baseline as
assessed by T2* MRI.
o Change in serum ferritin level from baseline over time
 Proportion of subjects receiving iron chelation therapy over time
2.2.5. Exploratory Endpoints

 Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-
cells) from baseline (pre-transfusion) over time
 Exploratory biomarker analysis of peripheral blood as compared to baseline over
time, including but not necessarily limited to expression of α-globin and non-α-globin
mRNA in circulating reticulocytes over time.
 Assessment of erythropoiesis in bone marrow analysis compared with baseline over
time
3. STUDY POPULATION
3.1. Number of Subjects

In the first part of the study, at least 2 adult non-β0/β0 subjects will be enrolled before expansion
to include β0/β0. After at least subjects ≥18 years old have been enrolled and have
of efficacy and safety data available following infusion of CTX001, the study may be
expanded to include subjects 12 to <18 years old. Approximately adult subjects will be dosed
with CTX001 before the conditioning and dosing of the first pediatric subject. After or more
TDT subjects have been enrolled and dosed with CTX001, the trial may be expanded to up to
45 subjects dosed.
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3.2. Subject Inclusion Criteria

study:
1. Subjects 12 to 35 years of age, inclusive, on the date of informed consent.
2. Subject (or their legally authorized representative or guardian) will sign and date an informed
consent form (ICF) and, where applicable, an assent form.
a. Documented homozygous β-thalassemia or compound heterozygous β-thalassemia
including β-thalassemia/hemoglobin E (HbE). Subjects can be enrolled based on historical
data, but a confirmation of the genotype using the study central laboratory will be required
before busulfan conditioning. The β0 and non β0 genotypes are defined using the HbVar
Database.
b. A history of at least 100 mL/kg/year or 10 units/year of packed RBC transfusions in the
prior 2 years before signing the consent or the last rescreening for patients going through re-
screening.
4. Karnofsky performance status of ≥80% for subjects ≥16 years of age. Lansky performance
status of ≥80% for subjects <16 years of age.
6. Access to detailed medical records on packed RBC transfusions, including volume or units of
packed RBCs and associated pre-transfusion Hb values, and in-patient hospitalizations, for at
least the 2 years prior to consent.
8. Male subjects of reproductive capacity must agree to use effective contraception from start of
mobilization through at least 6 months after CTX001 infusion (Section 3.6).
9. Willing and able to comply with scheduled visits, treatment plan, laboratory tests,
contraceptive guidelines, and other study procedures.
10. Willing to participate in an additional long-term follow-up study (Study VX18-CTX001-131)
after completion of this study.
3.3. Subject Exclusion Criteria

Subjects meeting any of the following criteria are not eligible for enrollment in the study:
1. An available 10/10 Human Leukocyte Antigen (HLA)-matched related donor.
2. Prior allo-HSCT.
3. Subjects with associated α-thalassemia and >1 alpha chain deletion, or alpha multiplications.
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4. Subjects with sickle cell β-thalassemia variant.

6. White blood cell (WBC) count <3 × 109/L or platelet count <50 × 109/L not related to
hypersplenism, per investigator judgment.
confound the results of the study or pose an additional risk in administering study drug to the
subject. This may include but is not limited to: immediate family member with a known family
cancer syndrome, history of relevant drug allergies; history of cardiovascular or central nervous
system disease; history or presence of clinically significant pathology; history of uncontrolled
seizure disorders, or history of psychiatric disorders.
a. Aspartate transaminase (AST), alanine transaminase (ALT) >3 x the upper limit of
normal (ULN), or direct bilirubin value >2.5 x the ULN, or:
c. History of cirrhosis or any evidence of bridging fibrosis, or active hepatitis on previous
liver biopsy.
d. Liver iron content (LIC) ≥15 mg/g on R2* MRI of liver.
11. A cardiac T2* <10 ms by MRI or left ventricular ejection fraction (LVEF) <45% by
echocardiogram.
13. Diffusing capacity of the lungs for carbon monoxide (DLco) <50% of predicted (corrected
for hemoglobin and/or alveolar volume).
15. Intolerance, contraindication, or known sensitivity to plerixafor, granulocyte colony
stimulating factor (G-CSF) products (e.g., filgrastim), or busulfan. Prior anaphylaxis with
excipients of CTX001 product (Dimethyl sulfoxide [DMSO], Dextran).
16. Positive for the presence of human immunodeficiency virus-1 (HIV-1) or human
immunodeficiency virus-2 (HIV-2) (positive antigen/antibody AND nucleic acid tests [NAT]),
hepatitis B virus (HBV) (positive hepatitis B core antibody [HBcAb] AND NAT tests), syphilis
[positive screening AND confirmatory tests], or hepatitis C virus (HCV; positive antibody
[HCAb] and NAT tests). Additional infectious disease markers should be obtained and tested as
required by the local authority for the collection and processing of cellular therapy products.
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These additional tests (e.g., HTLV-1, HTLV-2, malaria, tuberculosis, toxoplasmosis,

Typanosoma cruzi, or West Nile virus) will be evaluated to determine overall impact to the
patient and manufacturing of CTX001.
17. Participation in another clinical study with an investigational drug within 30 days of
screening.
18. An assessment by the investigator that the subject would not comply with the study
procedures outlined in the protocol.
3.4. Enrolled Subjects and Screen Failures

Enrolled subjects are defined as subjects who consent to participate in the clinical trial and
whose eligibility is confirmed (meet the inclusion/exclusion criteria).
Screen failures are defined as subjects who consent to participate in the clinical trial but do not
meet the eligibility criteria.
Rescreening and Repetition of Screening Assessments: Individuals who did not meet the
eligibility criteria (screen failure) can be rescreened up to 2 times. All screening tests should be
repeated to determine eligibility except for: genotyping (if performed during the initial
screening), R2* MRI of liver, T2* MRI of the heart (if performed within 6 months and
pulmonary function tests (PFTs) and ECHO if performed within 3 months), and infectious
disease markers (if performed within 60 days).
Repetition of individual screening assessment(s) that did not meet eligibility requirements is not
permitted with the following exceptions:
 If there is clear evidence of a laboratory error (e.g., hemolyzed sample) or equipment
malfunction, collection of a repeat sample for the appropriate laboratory test or assessment
may be permitted with the approval of the Medical Monitor.
 Individual laboratory results that are related to a temporary, reversible condition in the
opinion of the investigator may be retested once after the condition resolves or within 30
days, whichever is earlier.
If repeat values of the individual assessment(s) are within the eligibility criteria and completed
within the screening window, then the subject is eligible for the study. Rescreened participants
will keep the same participant number assigned during the initial screening process.
3.5. Waivers of Inclusion/Exclusion Criteria

Waivers will not be permitted in the study.
3.6. Contraception
Study participation requires a commitment to use 2 highly effective methods of birth control.
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Female subjects of childbearing potential must agree to use acceptable, highly effective methods
infusion.
Non-sterile male subjects of reproductive capacity who are or may become sexually active with
female partners of childbearing potential must agree to use acceptable, highly effective methods
of contraception to avoid fathering a child from start of mobilization through at least 6 months
Acceptable and highly effective methods of contraception for subjects and their partners are
listed below. If applicable, additional contraception requirements may need to be followed
according to local regulations and/or requirements.
• True abstinence from heterosexual intercourse for the subject, when this is in line with
the preferred and usual lifestyle of the subject. Periodic abstinence (e.g., calendar,
ovulation, symptothermal, post-ovulation methods) and withdrawal are not acceptable
methods of contraception.
• If the male is infertile (e.g. after bilateral orchiectomy) or pre-pubescent. Infertility may
be documented through examination of a semen specimen before mobilization.
Female Subjects
 Female bilateral tubal ligation performed at least 6 months previously.
 Female continuous use of an intrauterine device (non-hormone releasing or hormone
 Female combined (estrogen and progestogen-containing) or progestogen-only oral
hormonal contraception associated with inhibition of ovulation if successfully used for at
least 60 days before mobilization or with a second form of approved contraception for at
least 60 days after beginning hormonal contraception.
 Female subjects undergoing fertility preservation as described in Section 8.12 and who
are unable to use hormonal contraception during this time must use true abstinence
during the period of fertility preservation unless meeting another criterion listed as
waivers of contraception for the couple below.
Male Subjects
Acceptable contraceptive methods must be used from start of mobilization through at least
6 months after CTX001 infusion and include the following:
• True abstinence from heterosexual intercourse, when this is in line with the preferred and
usual lifestyle of the subject. Periodic abstinence (e.g., calendar, ovulation,
symptothermal, post-ovulation methods) and withdrawal are not acceptable methods of
contraception.
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contraception.
• Hormonal contraceptives, if successfully used for at least 60 days before start of
mobilization
Additional notes:
• Female condom cannot be used with male condom (as a double method of contraception)
due to risk of tearing.
• The use of birth control methods does not apply if the female partner has had a bilateral
oophorectomy, hysterectomy, or is postmenopausal (as defined below).
• Male subjects who are not sexually active at the time of screening must agree to follow
the contraceptive requirements of this study if they become sexually active with a partner
of the opposite sex.
• If over the course of the study the subject’s status changes and the subject does not meet
the criteria for waiving the contraception requirements, the subject must begin following
the contraceptive methods listed above.
• If applicable, additional contraception requirements may need to be followed according
to local regulations and/or requirements.
• Male subjects must not donate sperm after start of mobilization, throughout the study,
and for 6 months following CTX001 infusion.
• Unique situations that may not fall within the above specifications may be discussed with
the Sponsor’s medical monitor or designee on an individual basis.
• For subjects who have not yet gone through puberty or unable to provide oocytes/sperm
for fertility preservation, local SOPs and standards of care for tissue collection may be
offered to the patient at the discretion of the investigator.
Female Subjects of Non-Childbearing Potential:
Female subjects of non-childbearing potential will not be required to use contraception. To be
considered of non-childbearing potential, female subjects must meet at least 1 of the following
criteria:
• Postmenopausal: Female subjects who have been amenorrheic for at least 2 years and
have a serum follicle-stimulating hormone (FSH) level within the laboratory's reference
range for postmenopausal females
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inclusion/exclusion criteria have the option to undergo fertility preservation via cryopreservation
of oocyte or sperm, or gonadal tissue banking as appropriate per subject age and local practice,
(this may occur at any time after eligibility is confirmed and prior to initiation of busulfan
conditioning).
Stage 2: Mobilization, autologous CD34+ stem cell collection, CTX001 manufacture and
disposition
Before starting administration of plerixafor and G-CSF, subjects will be assessed by the study
investigator to confirm whether they are eligible to proceed with apheresis (as per local
guidelines).
After eligibility has been confirmed, each subject will undergo stem cell mobilization with a
combination of G-CSF products (e.g. filgrastim) and plerixafor. Peripheral blood mononuclear
cells (PBMCs) will be collected by apheresis. Subjects will undergo apheresis for 2 or
3 consecutive days of apheresis to collect CD34+ hHSPC. The targeted CD34+ cell collection is
at least 15 x 106 CD34+ cells/kg for manufacturing of CTX001. An additional 2 x 106 CD34+
cells/kg will be collected as backup for rescue therapy in an event of non-engraftment with
CTX001. Cell collection intended for manufacturing will only occur on Days 1 and 2 of
apheresis. On these days, collected cells intended for manufacturing will be shipped daily at 2 to
8oC to the manufacturing facility. Day 3 of apheresis is reserved for collection of backup cells
ONLY. These cells will not undergo the editing manufacturing process and will be
cryopreserved at the site. this must occur before proceeding to Stage 3 busulfan conditioning.
Backup cells may also be collected on Day 2 of apheresis, ONLY after enough cells have been
collected for manufacturing.
If sufficient numbers of cells for CTX001 manufacturing and backup are not obtained (as
determined based on discussion between the Sponsor and investigator), up to 2 additional
mobilization and apheresis cycles will be allowed to collect additional cells. The additional
mobilization and apheresis cycle may be performed at least 14 days after the first day of the prior
mobilization cycle and no more than 60 days after the end of the prior cycle.
See Section 6 (Treatment Plan) for further description of mobilization, apheresis, busulfan
conditioning, infusion, and shipment to the manufacturing facility.
Stage 3: Myeloablative conditioning (Stage 3A), and infusion of CTX001 (Day 1, Stage 3B)
Stage 3A – Myeloablative conditioning: After the CTX001 product is received at the site and it
has been confirmed that the backup cells remain available and in suitable condition to be
administered if needed, the subject will be hospitalized to undergo myeloablative conditioning
with busulfan.
Busulfan will be administered intravenously (IV) daily at a starting dose of 3.2 mg/kg/day (based
on weight collected 3-7 days prior to initiation of busulfan) for 4 consecutive days. Once-daily
dosing is the preferred schedule; however, the busulfan dosing regimen may be adjusted to be
given as 0.8 mg/kg every 6 hours (Q6H) for 4 consecutive days, per the site’s standard practice.
The dose of busulfan may be adjusted based on the pharmacokinetics (PK) of the first busulfan
dose to maintain appropriate levels for myeloablation. The average target area under the curve
(AUC) for subjects receiving a starting dose of 3.2 mg/kg/day for 4 days is 5000 µM*min
(range: 4500 to 5500 µM*min); equivalent to a target cumulative busulfan exposure of
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90 mg x hr/L (range 80-100 mg x hr/L). The mean target AUC for subjects administered
busulfan Q6H for 4 days is 1125 µM*min (range: 900 to 1350 µM*min). For subjects less than
34 kg, institutional and/or regional practice for busulfan dosing may be used.
Clinical sites may use a test dose of busulfan within 30 days (consistent with institutional
guidelines) prior to initiation of busulfan to pre-determine busulfan dose.
Infusion of busulfan should not take place if:
 CTX001 integrity has been compromised in some way and is no longer suitable for
infusion (e.g., damage to the product container in transit to the clinical site).
 The subject has any clinical condition that, in the opinion of the investigator, would put
the subject at unacceptable risk for transplantation.
In the event that full dose of busulfan chemotherapy has not been administered, the investigator
should immediately inform the medical monitor.
Stage 3B - CTX001 infusion: Infusion of CTX001 will occur at a minimum of 48 hours
following the completion of busulfan infusion and at a maximum of 7 days after the completion
of busulfan infusion.
On Day 1, each vial will be administered no more than 20 minutes from time of thaw. The entire
dose of CTX001, which at a minimum will be 3.0 × 106 CD34+ cells/kg, will be infused through
a central venous catheter. All vial(s) containing CTX001 should be infused.
If the investigator suspects that CTX001 integrity has been compromised in some way and is no
longer suitable for infusion (e.g., damage to the product container in transit the clinical site), the
investigator should not infuse CTX001 and immediately contact the medical monitor.
Stage 4: Follow-up, through engraftment and up to 24 months after CTX001 infusion
Stage 4A - Post-infusion until hospital discharge (post-engraftment): Subjects will be followed
daily in the transplant unit and receive supportive care according to standard practices for
subjects undergoing hematopoietic stem cell transplant (HSCT). Subjects will be monitored for
AEs and engraftment. Packed RBC and platelet transfusions will be given to subjects per
standard practices/investigator judgment for subjects undergoing HSCT. Subjects will be
discharged from the transplant unit upon neutrophil engraftment (defined as ANC ≥500/µL for
3 measurements on 3 consecutive days) and stabilization of major medical issues as per local
hospital guidelines and/or investigator judgment. Subjects may receive G-CSF if no engraftment
is apparent by Day 21, after a discussion between the investigator and study medical monitor.
If engraftment is not achieved within 42 days after CTX001 infusion, the subject will receive the
backup CD34+ stem cells. In addition, the investigator may decide to administer the backup cells
before Day 42 if clinically indicated.
Stage 4B - Post-engraftment Follow-up: Stage 4B starts after subjects have successfully
engrafted, are clinically stable, and have been discharged from the transplant unit. Subjects will
be followed for 24 months after CTX001 infusion with physical exams, laboratory and imaging
assessments, and AE evaluations. Subjects will be allowed to restart iron chelation
approximately 1 month after CTX001 infusion according to the site’s management guidelines
and/or investigator judgment.
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Following engraftment, transfusions of packed RBCs should be avoided for Hb ≥9 g/dL, unless
medically indicated (e.g., symptomatic anemia or as a requirement for surgery). It is
recommended that subjects should receive packed RBC transfusions for Hb <7.0 g/dL, while
medical judgement is advised to transfuse for Hb levels of 7-9 g/dL based on a subject’s clinical
needs.
Following receipt of busulfan conditioning and CTX001, subjects should undergo at least yearly
PEs, and receive screening for malignancy based on appropriate country-specific cancer
guidelines and subject medical history. Subjects should also undergo appropriate malignancy
evaluation if they have unexplained symptoms, signs, or laboratory abnormalities that could be
related to an underlying malignancy. Examples include unexplained weight loss or fever,
lymphadenopathy, and abnormal blood counts. The medical monitor should be notified if a
malignancy is found.
All subjects infused with CTX001 will be asked to enroll in a long-term follow-up study
(Study VX18-CTX001-131) following completion or withdrawal/discontinuation.
4.2. Study Duration

The duration of Stage 1 will be approximately 1 to 3 months; Stage 2 approximately
2 to 3 months; Stage 3 approximately 1 month; Stage 4 approximately 2 years.
Subjects will be followed on study for a total of approximately 2 years after CTX001 infusion.
follow-up study (Study VX18-CTX001-131) following completion of or
withdrawal/discontinuation from this study.
4.3. Study Enrollment Plan

The first 2 subjects enrolled in the study will be adults with a non-β0/β0 genotype and will receive
CTX001 in a staggered manner to ensure that there is successful engraftment of the first subject
before treating the second subject in the study. The second subject will not undergo
myeloablation until the first subject achieves neutrophil engraftment (ANC ≥500/µL for 3
measurements on 3 consecutive days) and the available engraftment and safety data have been
reviewed by the DMC and at least 30 days after infusion of CTX001 to the first subject. If the
second subject infused with CTX001 has not experienced Grade ≥3 AEs other than those
typically associated with busulfan conditioning or autologous transplant procedure through
engraftment or 30 days post infusion (whichever is longer), the remaining subjects, which may
include subjects with the β0/β0 genotype, can undergo conditioning and CTX001 infusion
concurrently. In the event that the second subject infused with CTX001 experiences Grade ≥3
AEs other than those typically associated with busulfan conditioning or autologous transplant
procedure, a DMC meeting will be convened and data will be reviewed before the remaining
subjects can undergo conditioning and CTX001 infusion. In this case, a substantial amendment
to the clinical trial application (CTA) will be submitted before the remaining subjects can
undergo conditioning and CTX001 infusion. All steps that precede busulfan myeloablation such
as consent, screening, stem cell collection, and manufacturing may proceed concurrently without
staggering subjects.
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In addition, a DMC meeting will be convened after at least subjects ≥18 years old have been
enrolled and have of efficacy and safety data following infusion of CTX001 to
support expansion to include subjects 12 to <18 years old. Approximately adult subjects will
be dosed with CTX001 before the conditioning and dosing of the first pediatric subject.
Further, the DMC will review safety and efficacy data after at least subjects have received
CTX001 infusion in order to recommend whether or not to expand the study to dose up to 45
subjects.
5. STUDY STOPPING RULES
5.1. Enrollment Suspension Criteria

The investigator will immediately notify the medical monitor of any of the following:
 Fatal or life-threatening SAEs
 SAEs related to CTX001
 Engraftment failures
 Malignancies
 Failure to manufacture the target cell dose of CTX001
Any SAEs will also be reported to Vertex Global Patient Safety in accordance with
Section 9.1.2.
Enrollment will be temporarily suspended for any of the following safety reasons:
 Death (non-accidental) on study after CTX001 infusion.
 Detection of leukemia, myelodysplastic disease, or myeloproliferative disorder in a
subject who has received CTX001
 Failure in two subjects to achieve neutrophil engraftment after infusion of CTX001, as
defined by lack of engraftment within 42 days after infusion or administration of backup
cells.
 Any serious adverse event (SAE) at least possibly related to CTX001, but not related to
busulfan in 3 subjects, with the exception of
o Asymptomatic elevations in amylase or lipase
o Grade 3 elevation in ALT, aspartate transaminase (AST), alkaline phosphatase,
gamma-glutamyl transferase (GGT), or bilirubin that resolve to baseline or within
normal range in 10 days
o Grade 3 infusion reaction (including those reported as a hypersensitivity/allergic
event and/or signs or symptoms of infusion reactions) that resolves in 2 days
o Grade 3 or 4 fever associated with engraftment that resolves in 4 days
Enrollment will be temporarily suspended for any of the following lack of efficacy reasons:
 HbF <1g/dL at 9 months in 4 subjects and ≥80% of those enrolled
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 The proportion of alleles with intended genetic modification present in peripheral blood
leukocytes is <10% in 3 of the first 12 subjects at 2 consecutive timepoints after
neutrophil engraftment
If there is any such event(s), enrollment will be temporarily suspended, and all available data
will be reviewed. After evaluation and following a review of the data with the DMC, the SC and
Sponsor may decide to restart enrollment in the study, amend the study, or permanently suspend
reviewed by the DMC. Based on the review of data and the recommendations of the DMC, the
SC and Sponsor may decide to restart enrollment in the study, amend the study, or permanently
suspend enrollment and remove all subjects who have not received CTX001 from the study. A
substantial amendment will be submitted before resuming enrollment.
In the event enrollment is permanently suspended, subjects who are already enrolled in the study
followed up or transitioned to a long-term safety follow-up study (Study VX18-CTX001-131).
unedited CD34+ cells to the subject will be up to the investigator’s discretion after discussion of
the potential risks with the subject. The investigator should inform the medical monitor of the
decision before infusion of cellular product.
If enrollment is permanently suspended, the Sponsor will immediately inform all appropriate
parties including Principal Investigators (PIs), Ethics Committees (EC), Institutional Review
Boards (IRB) and competent authorities.
5.2. Stopping Rules

 Any medical condition that, in the opinion of the investigator, would put the subject at
risk for continuing study-related procedures or follow-up.
 If a subject is found not to have met eligibility criteria or has an important protocol
deviation before the start of busulfan conditioning that, in the opinion of the investigator,
would put the subject at risk for continuing study-related procedures or follow-up
 Subject becomes pregnant or begins breastfeeding prior to start of busulfan conditioning
 Inability or anticipated inability to manufacture the target cell dose of CTX001
5.3. End of Study Definition

The end of study is defined as the last scheduled visit (or contact) of the last subject.
The Sponsor will notify all applicable regulatory agencies in accordance with local requirements
when the study has ended.
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5.4. Sponsor’s Termination of Study

At any time, the Sponsor may terminate this study in its entirety or may terminate this study at
any particular site. In addition, for reasonable cause, either the investigators or their IRBs/IECs
may terminate the study at their center.
Conditions that may lead to reasonable cause and warrant termination include, but are not limited
to:
 Subject or investigator noncompliance
 Unsatisfactory subject enrollment
 Lack of adherence to protocol procedures
 Lack of evaluable and/or complete data
 Potentially unacceptable risk to study subjects
 Decision to modify drug development plan
 Decision by the FDA or other regulatory authority
Written notification that includes the reason for the clinical study termination is required.
on a long-term follow-up study (Study VX18-CTX001-131).
6. TREATMENT PLAN
6.1. Description of CTX001
CTX001 product is composed of autologous CD34+ hHSPCs modified with CRISPR-Cas9 at the
erythroid lineage-specific enhancer of the BCL11A gene. CTX001 will be formulated in
CryoStor CS5 medium which contains 5% DMSO and Dextran 40. Subjects will receive the
entire dose of CTX001, which will be at a minimum of 3.0 × 106 CD34+ cells/kg, through a
central venous catheter.
6.2. Mobilization and Apheresis

6.2.1. Mobilization
Before starting administration of plerixafor and filgrastim, subjects will be assessed by the study
investigator to confirm whether they are eligible to proceed with apheresis (as per local
guidelines). Examples of ineligibility include hemodynamic instability, positive infectious
serologies, or active infection. If a subject is deemed to not be eligible for mobilization and
apheresis, this procedure can be delayed for up to 2 months. Thereafter, the subject will be
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6.2.2. Apheresis Procedure

CD34+ hHSPC will be collected per clinical site SOPs for up to 3 consecutive days. Subjects will
undergo apheresis for 2 consecutive days to collect CD34+ hHSPC for CTX001 manufacturing.
The targeted CD34+ cell collection is at least 15 x 106 CD34+ cells/kg in order to achieve a
minimum target dose of 3 × 106 CD34+ cells/kg. On these days collected cells intended for
manufacturing will be shipped same-day at 2 to 8˚C to the manufacturing facility. An additional
2 x 106 CD34+ cells/kg will be collected as backup for rescue therapy in an event of non-
engraftment with CTX001. These cells will not undergo the editing manufacturing process and
will be cryopreserved at the site: this must occur prior to proceeding to Stage 3 busulfan
conditioning. Backup cell collection may occur on Day 3 immediately following the consecutive
CTX001 manufacturing cell collections or may be postponed to an additional mobilization and
apheresis cycle.
Cell collection intended for manufacturing will only occur on Days 1 and 2 of apheresis. Day 3
of apheresis is reserved for collection of backup cells ONLY. Backup cells may also be collected
on Day 2 of apheresis, ONLY after enough cells have been collected for manufacturing.
If the first mobilization and apheresis cycle does not yield enough cells for both the minimum
CTX001 product and safety backup, up to 2 additional mobilization and apheresis cycles will be
allowed to collect additional cells. The additional mobilization and apheresis cycle may be
performed at least 14 days after the first day of initial mobilization cycle and no more than
60 days after the end of the prior cycle. Based on the number of CD34+ cells received at the
manufacturing site and the manufactured CTX001 dose, the medical monitor will inform the site
if an additional mobilization and apheresis cycle will be necessary. Study investigator should
contact the medical monitor if the minimal cell dose for manufacturing or backup is not reached
or if the subject requires an additional mobilization cycle.
Please refer to the study reference manual for further collection details and shipment instructions.
Any AEs associated with apheresis procedure should be managed as per site’s standard
guidelines.
6.3. Shipment to Manufacturing Facility

At the end of the first and second day of apheresis, collected cells will be shipped at 2-8°C to the
manufacturing facility. Cells collected for a backup dose will be cryopreserved and stored at the
study site. Additional details and instructions on shipment and receipt are included in the study
reference manual.
6.4. Manufacture of CTX001

Manufacturing of the cell product inclusive of quality release testing is expected to take
approximately . Following arrival of the cells at the manufacturing facility, CD34+ cells
are isolated and incubated in culture media. The cells are then recovered, pooled, and treated
with CRISPR-Cas9 gene-editing reagents. The cells are again allowed to recover in culture
medium before harvest, formulation, sampling for quality control (QC) testing, filling, and
cryopreservation in the vapor phase of liquid nitrogen (≤ -135°C). QC testing and batch release
should normally be completed in . Product will then be shipped at ≤ -135°C to the
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clinical site in one or more 20 mL vials. All manufacturing procedures are carried out in Good
Manufacturing Practice (GMP) cleanrooms.
6.5. Shipment of CTX001 Product to Treatment Site

CTX001 (packaged in vial(s)) will be shipped cryopreserved to the clinical sites in sealed dry
nitrogen shippers (≤135°C) validated for at least 10 days. On receipt, package integrity will be
checked and the cellular product label(s) will be confirmed against the donor’s unique identifier.
Vial(s) will be labeled for “autologous use only” with donor identification, batch number, and
expiry date in accordance with local regulatory requirements. Additional details and instructions
are included in the study reference manual.
6.6. Storage of CTX001 Product

CTX001 will be stored at the site in the frozen state at a temperature of ≤ -135°C until just prior
to the scheduled infusion. Additional details and instructions are included in the study reference
manual.
6.7. Conditioning: Busulfan Administration

Busulfan eligibility should be reconfirmed as per Table 4 and per site’s guidelines and
investigator judgment. If the subject is found not to be appropriate to undergo busulfan
conditioning and CTX001 infusion, they may be re-assessed 2 weeks later if the ineligibility is
due to an acute process that will resolve. Otherwise subjects should be withdrawn from the study
and undergo end of study visit. If 3 months pass from the time of screening procedures to the
planned start of myeloablation with busulfan, PFTs and echocardiogram should be performed
within 30 days prior to busulfan administration to confirm eligibility. If 6 months pass from the
time of screening procedures to the planned start of myeloablation with busulfan, MRIs will also
be repeated to confirm eligibility.
Busulfan conditioning should only start once CTX001 is received at site and results of
genotyping for thalassemia (alpha and HBB loci) have been confirmed. Busulfan will be
administered IV daily at a starting dose of 3.2 mg/kg/day for 4 consecutive days (based on
weight collected within 3-7 days prior to the first day of busulfan administration). Once-daily
dosing is the preferred schedule, but the busulfan dose regimen may be adjusted to be given as
0.8 mg/kg every 6 hours (Q6H) for 4 consecutive days, per the site’s standard practice. A test
dose of busulfan may be performed within 30 days (consistent with institutional guidelines) prior
to initiation of busulfan.
The dose of busulfan may be adjusted based upon first dose busulfan PK in order to maintain
appropriate levels for myeloablation. The average target AUC for subjects at a starting dose of
3.2 mg/kg/day for 4 days is 5000 µM*min (range: 4500 to 5500); equivalent to target cumulative
busulfan exposure of 90 mg x hr/L [range 80 to 100 mg x hr/L]). The AUC for subjects
administered busulfan- Q6H for 4 days is 1125 µM*min (range: 900 to 1350). Dose adjustment
will be performed for obese subjects or for subjects with renal or hepatic impairment per local
label and standard of care. For subjects less than 34 kg, institutional and/or regional practice for
busulfan dosing may be used.
Refer to Appendix 1 for busulfan PK collection schedule.
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During busulfan conditioning, anti-seizure prophylaxis and other supportive measures should be
instituted as per hospital guidelines.
 CTX001 integrity has been compromised in some way and is no longer suitable for
 The subject has any clinical condition which, in the opinion of the investigator, would put
6.8. CTX001 Infusion Procedures, Dose, and Administration

CTX001 will be formulated in CryoStor CS5 medium which contains 5% DMSO and
Dextran-40. Histamine release associated with DMSO can result in symptoms such as, adverse
effects including nausea, vomiting, diarrhea, flushing, fevers, chills, headache, dyspnea, rashes,
bronchospasm, anaphylaxis, vasodilation and hypotension, and mental status changes. Given the
risk of these AEs, subjects will be pre-medicated with an antihistamine (diphenhydramine
hydrochloride) and an antipyretic (acetaminophen or paracetamol) before dosing with CTX001
as per institutional guidelines. These medications may be repeated every 3-4 hours as needed as
per institutional guidelines and investigator judgment.
Emergency medical equipment should be available during the infusion in the event a subject has
an allergic response, severe hypotensive crisis, or any other infusion-related reaction.
If multiple weights have been taken during the 5 days prior to first day of mobilization, then the
weight recorded closest to time of mobilization should be used for calculation of the cell dose.
Refer to Table 4 for full dosing schedule.
The single dose of CTX001 will be given at least 48 hours and within 7 days after the last
busulfan dose. CTX001 vial(s) should be thawed just prior to the scheduled infusion as per local
site SOPs and infused within 20 minutes of thaw. Detailed instructions for the thaw and infusion
of cells are in the study reference manual. Hospital guidelines will be used to maintain chain of
custody for CTX001 from the stem cell lab to the subject. Prior to infusion, local procedures
should be followed regarding the verification of subject identity and product details to ensure a
match as well as integrity of the product.
Subjects will receive CTX001 on Day 1 via infusion through a central venous catheter. All
vial(s) containing CTX001 should be infused.
All procedures involving CTX001 must be performed using aseptic techniques by trained
personnel according to the SOP at the clinical site.
Infusion of CTX001 should not take place if CTX001 integrity has been compromised in some
way and is no longer suitable for infusion (e.g., damage to the product container in transit the
clinical site).
In the unlikely event that CTX001 infusion does not occur, subjects should receive the backup
CD34+ stem cells.
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G-CSF should not be administered following CTX001 infusion without the explicit approval of
the medical monitor.
6.9. Post-CTX001 Infusion Infection Prophylaxis and Surveillance

Following CTX001 infusion, subjects will undergo infectious surveillance and prophylaxis
(bacterial, viral, fungal) as per local guidelines for HSCT and investigator judgment.
In the event that the subject develops sepsis or systemic bacteremia following CTX001 infusion,
appropriate cultures and medical management will be initiated.
Broad spectrum antibiotic treatment for febrile neutropenia and other supportive measures will
be administered as per local hospital guidelines for autologous stem cell transplantation and
Transfusion of packed RBC following CTX001 is specified in Section 6.11.3.
If engraftment does not occur by Day 21 post-CTX001 infusion, G-CSF may be administered
until the ANC is ≥500/uL for 3 measurements on 3 consecutive days. The use of G-CSF post-
CTX001 infusion should be discussed with the study medical monitor prior to initiation.
6.10. Screening for Malignancy

PEs and receive screening for malignancy based on appropriate country-specific cancer
guidelines and subject medical history. Subjects should also undergo appropriate malignancy
evaluation if they have unexplained symptoms, signs, or laboratory abnormalities that could be
related to an underlying malignancy. Examples include unexplained weight loss or fever,
lymphadenopathy, and abnormal blood counts. The medical monitor should be notified if a
malignancy is found, and any new malignancy should be reported as an SAE.
6.11. Prior and Concomitant Treatments and Procedures

6.11.1. Prior medications
All medication taken within 30 days before signing of the ICF (and, if applicable, the assent
form) will be recorded.
Retrospective information on packed RBC transfusions and iron chelation will be recorded from
24 months prior to date of consent.
6.11.2. Venous Access

A central venous catheter will be used for administration of the conditioning regimen and
infusion of CTX001. A central venous catheter may also be used for apheresis, and for clinical
care of the subject following CTX001 infusion as per investigator judgment.
6.11.3. Transfusions
Prior to start of apheresis procedure and at least 30 days prior to planned initiation of busulfan
conditioning, subjects should be transfused to achieve goal of Hb ≥11 g/dL. This is done to
suppress ineffective erythropoiesis and allow for a more successful engraftment. During
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hospitalization for busulfan conditioning and CTX001 infusion subjects should be supported
with packed RBC and platelet transfusions as per standard practices for subjects undergoing
HSCT.
During the 24-month follow-up period after infusion of CTX001, it is recommended that subjects
receive packed RBCs for Hb ≤7g/dL and for clinical symptoms requiring transfusion. Reason for
all transfusions should be documented. Transfusions should be avoided for Hb≥9 g/dL, unless
considered clinically important (e.g., surgery).
All blood products will be filtered and irradiated as per hospital guidelines.
6.11.4. Iron Chelation

All iron chelation drugs must be discontinued at least 7 days prior to starting myeloablative
Iron chelation with deferasirox, deferoxamine, or deferiprone should not be started until at least 1
month following CTX001 infusion to allow for stable hematopoietic recovery and avoid
potential myelosuppressive effects. Subjects should be evaluated regularly to determine whether
chelation is required. Chelators should be administered according to transfusion requirements
and iron overload level when choosing the initial dose, escalation and interruption rules as per
institutional guidelines.
Potential reasons for discontinuing iron chelation are:
 Serum ferritin <1000 μg/L
 LIC<7 mg/g
 Transfusion independence and able to undergo phlebotomy as an alternative to iron
chelation
Phlebotomy may be used instead of chelation for subjects with Hb persistently ≥11 g/dL and
who are transfusion independent.
6.11.5. Prohibited medications

There are no prohibited medications.
6.12. CTX001 Accountability

compliance with applicable regulations and standard hospital procedures. Destruction will be
adequately documented.
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6.13. Treatment Compliance

Treatment will be administered per protocol in the clinic by trained study personnel.
6.14. Allelic Editing of CTX001 Drug Product

The proportion of alleles with intended genetic modification present in each batch of CTX001
will be assessed.
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7. VISIT EVALUATION SCHEDULE

The procedures and assessments that will be conducted during this study are described in this
section in narrative form starting in Section 8, and presented in separate tables for
Screening/Stage 1 (Table 3), Autologous CD34+ stem cell collection/Stage 2 (Table 4),
Myeloablative conditioning and infusion of CTX001/Stages 3A/3B (Table 5), and Post-Infusion
through Follow-up/Stage 4 (Table 6).
Table 3: Schedule of Events: Screening (Stage 1)

Screening
Procedure (up to 8 weeks)
Informed consent and, where applicable, assent X
PRO assessment (EQ-5D-5L and FACT-BMT for subjects ≥18 years old at Screening; X
EQ-5D-Y and PedsQL for subjects <18 years old at Screening)g
Demographics X
Medical historya X
Blood for thalassemia genotypingb (central lab) X
Physical examinationc X
Performance statusc X
Height and weight X
Vital signsd X
12-Lead ECG X
Serum chemistrye (local lab) X
Coagulatione (local lab) X
Hematologye (local lab) X
Serum pregnancy test for females of childbearing potential (local lab; See Section 8.9) X
Infectious disease marker testinge (local lab) X
Urinalysise (local lab) X
Liver MRI (R2*) (central lab) i X
Cardiac MRI (T2*) (central lab) i X
Echocardiogram X
Lung diffusing capacity for carbon monoxide (DLco) [corrected] X
Adverse event assessment (continuous from ICF and assent signing) Xh
Prior and concomitant medications (continuous from ICF and assent signing) X
a
Medical history including blood transfusion records, chelation and hospitalizations for at least 2 years before consent will be collected, as
well as thalassemia history including genotype (alpha and HBB loci and associated traits [e.g. Xmn1]), age of diagnosis, age of first
transfusion and age of initiation of chelation therapy (Section 8.1).
b
All subjects will be genotyped for thalassemia (alpha and HBB loci) irrespective of prior documentation of genotyping. Results from central
lab genotyping will be required before busulfan conditioning begins.
c
Physical examination including spleen assessment. The subject will be instructed to rest for at least 5 minutes before vital signs are
assessed. Performance status will be assessed using the Karnofsky Performance Status Scale for subjects ≥16 years old and the Lansky
Performance Status Scale for subjects <16 years old.
d
e
See Section 8.9.
f
Liver biopsy should only be performed after all other assessments have been done to assess eligibility. Subjects will undergo liver biopsy
when it is considered appropriate by the investigator and according to institutional standards.
g
PRO assessment should be performed as the first assessment after obtaining informed consent. A parent proxy version (EQ-5D-Y proxy
version) is available for subjects who are unable to complete the questionnaire themselves.
h
Section 9.1 and Table 8
i
Section 8.7
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Table 4: Schedule of Events: Autologous CD34+ Stem Cell Apheresis (Stage 2)

Procedure Mobilization Days of Apheresis
After Study Day -5 to Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Eligibility Day -1a (if
Confirmation (prior needed)
to mobilization
start)a
Iron studiesb (local lab) X
Immunological testing (local lab)b X
Allelic editing studies (central lab; % edited cells – baseline)c X
Hemoglobin fractionation (central lab)c X
F-cell distribution (central lab, flow cytometry)c X
α-globin and non-α-globin mRNA (central lab)c X
Exploratory research studies and assay development (central lab)c X
Bone marrow aspirate (local and central lab)d X
Sperm or oocyte banking, if requested by subject or their legally authorized Xe
representative
Infectious disease marker testing repeated Xf
Placement of central venous catheter for apheresis (if needed)g X
Hemolysis/erythropoiesis markers (local lab)b X
Mini-Mental State Examination (subjects ≥18 years old at time of
assessment) or Wechsler Abbreviated Scale of Intelligence (subjects 12 to X
<18 years old at time of assessment)
Eligibility for mobilization/apheresis re-confirmed X
Serum pregnancy test (females of childbearing potential only) Xh
Transfusion regimen (if required)i X
Physical examination j X X X X
Performance statusj X X X X
Vital signsk X X X X X X X
Height (for subjects <18 years at Screening) X
Weight Xl
Hematology (local lab) X X Xm Xm Xm
Serum chemistry (local lab) X Xm Xm Xm
Coagulation (local lab) X Xm Xm Xm
G-CSF administration (e.g. filgrastim) X X X X X Xn
Plerixafor administration X X Xo
Peripheral blood CD34+ count (local lab)q X Xm Xm Xm
CD34+ stem cell apheresis for CTX001 and backup cellsr X X X
Shipment of CD34+ stem cells to central manufacturer X X
Adverse event assessment Continuous from ICF and assent signingp
Prior and concomitant medications Continuous from ICF and assent signing
a
Assessments may be performed over multiple days or visits. If more than 1 apheresis cycle is required, only infectious disease marker testing must be repeated at this visit.
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b
See Section 8.9.
c
Samples drawn for the central lab should be performed immediately prior to a scheduled transfusion. See Section 8.10 and 8.11.
d
Bone marrow assessment of ineffective erythropoiesis (including myeloid:erythroid ratio) and iron stain. Central assessment for baseline testing of exploratory biomarkers.
e
Sperm or oocyte banking can be performed any time before busulfan conditioning after eligibility is confirmed. Other gonadal tissue banking may be done as appropriate per subject age and local
site practice. See Section 8.12.
f
Repeat IDMs if screening collection is more than 28 days prior to the planned apheresis start date. Collection should be no fewer than 10 days prior to the planned apheresis date.
g
As per site’s standard guidelines. Subject must be hemodynamically stable and have no active infection as per investigator judgment.
h
Pregnancy test should be negative prior to starting mobilization and must be performed within 3 days prior to start of mobilization.
i
Goal hemoglobin ≥11 g/dL prior to mobilization start.
j
Physical exam and performance status should be performed within 5 days prior to the first day of mobilization and on every day of apheresis prior to starting the collection. Physical examination
Includes assessment of spleen size.
k
Includes blood pressure (systolic and diastolic), temperature, pulse rate, respiration rate, and pulse oximetry. The subject will be instructed to rest for at least 5 minutes before vital signs are
assessed.
l
Weight taken within 5 days prior to the start of mobilization will be the weight used for plerixafor, G-CSF, and CTX001 dose calculations.
m
Labs to be performed each morning immediately prior to start of apheresis on Days 5-7. See Section 8.9.
n
For subjects with spleen, evening dose of G-CSF administered only if an apheresis collection is planned for Day 7.
o
Only if third day of apheresis is planned.
p
q
CD34+ counts are measured before the first plerixafor dose and immediately before each apheresis
r
Section 4.1
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Table 5: Schedule of Events: Myeloablative Conditioning and Infusion of CTX001

(Stages 3A/3B)
Procedure Pre-conditioning Conditioning Infusion
Within 30 days 3 to 7 days prior to Day -5 to -2 Day 1
prior to initiation of
initiation of busulfana
busulfana
Transfusion regimenb X
Serum pregnancy test (females of childbearing Xc
potential only)
Physical examination X X X
Performance status X X
Vital signsd X X Xe
Weight X
12-lead ECG X Xf
Stop iron chelationg Xg
Hematology (local lab) X X Xh
Serum chemistry (local lab) X X Xh
Eligibility for myeloablation re-confirmed Xi Xj
Confirm rescue cells are cryopreserved at the site X
Confirm CTX001 has been received at the site X
prior to initiating busulfan
Test dose of busulfan to determine optimal X
busulfan dose (optional)
Anti-seizure prophylaxis Xk
IV busulfan administration X
Busulfan pharmacokinetics Xl
IV Infusion of CTX001 (Section 6.8) X
Adverse event assessment Continuous from ICF and assent signingm
Prior and concomitant medications Continuous from ICF and assent signing
a
b
Maintain hemoglobin ≥ 11 g/dL at least 30 days prior to starting busulfan.
c
Pregnancy test must be negative within 5 days prior to starting busulfan.
d
The subject will be instructed to rest for at least 5 minutes before vital signs are assessed.
e
Measure vital signs prior to infusion and every 30 minutes after infusion up to 2 hours. Includes blood pressure (systolic and diastolic),
temperature, pulse rate, respiration rate, and pulse oximetry.
f
ECG performed prior to CTX001 infusion.
g
Iron chelation needs to be stopped at least 7 days prior to start of busulfan conditioning.
h
Collect labs prior to infusion on Day 1. See Section 8.9.
i
If screening and pre-conditioning visits are ≥3 months apart, pulmonary function tests and echocardiogram will also be repeated to
confirm eligibility, and MRIs will also be repeated to confirm eligibility if screening and pre-conditioning visits are ≥6 months apart
j
Review most recent assessments (within 7 days prior to conditioning: CBC+ differential, serum chemistry, liver function tests, ECG,
physical examination, performance status and pregnancy test results and additional tests) as per site’s standard guidelines for autologous
stem cell transplant.)
k
Initiate anti-seizure prophylaxis at least 12 hours before first dose of busulfan and continue until 24 hours after completion of the full
busulfan administration (as per local guidelines).
l
Blood sample will be collected for pharmacokinetic analysis for busulfan. If PK analysis is not available locally for daily results, perform
analysis on first and third day of dosing. See Appendix 1.
m
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Table 6: Schedule of Events: Post-infusion through Follow-up (Stage 4)

Daily Follow-upa
Procedure from D+30b D+60 D+90 D+120 D+150 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 ETFc
M1 M2 M3 M4 M5 M6 M9 M12 M15 M18 M21 M24
until
(±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±14d) (±30d) (±30d) (±30d)
Discharge
Vital signsd X X X X X X X X X X X X X X
Height and weight (for subjects X X X
<18 years old at Screening)
Hematology (local lab)e X X X X X X X X X X X X X X
Serum chemistry (local lab)e X X X X X X X X X X X X X X
Coagulation (local lab)e Xf X X X X X X X X X X X X X
Hemolysis/erythropoiesis X X X X X X X X X X
markers (local lab)e
Ferritin (local lab)e X X X X X X X X X X X X X
Physical examination X X X X X X X X X X X X X X
Performance status k X X X X X X X X X X X X X X
Immunological testing (local X X X X X
lab)e
Allelic editing studies (central Xl X X X X X X X X X X
lab; % edited cells)g
Hemoglobin fractionation X X X X X X X X X X X X X
(central lab)g
F-cell distribution (central lab)g X X X X X X X X X X X X X
α-globin and non-α-globin X X X X X X X
mRNA (central lab)g
Bone marrow aspirate (local X X X X
and central lab)h
Exploratory research studies X X X X X X
and assay developmentg
Liver MRI (R2*) (central lab) m X X X
Lung diffusing capacity for X X X
carbon monoxide (DLco)
[corrected]
Cardiac MRI (T2*) (central X X X
lab) m
12-lead ECG X X X
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Daily Follow-upa
Procedure from D+30b D+60 D+90 D+120 D+150 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 ETFc
M1 M2 M3 M4 M5 M6 M9 M12 M15 M18 M21 M24
until
(±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±14d) (±30d) (±30d) (±30d)
Discharge
PRO assessment (EQ-5D-5L X X X X X X X X X X X
and FACT-BMT for subjects
≥18 years old at Screening;
EQ-5D-Y and PedsQL for
subjects <18 years old at
Screening)i
Adverse event collection Continuous from ICF and assent signingj
Prior and concomitant Continuous from ICF and assent signing
medication
a
b
. Month (M) is defined as 30 days. If Day 30 occurs while the subject is still hospitalized, the daily assessments required prior to discharge should be done in addition to the Day 30 assessments
c
Early Termination of Follow-Up.
d
Includes blood pressure (systolic and diastolic), temperature, pulse rate, respiration rate, and pulse oximetry. The subject will be instructed to rest for at least 5 minutes before vital signs are
assessed.
e
See Section 8.9.
f
Weekly until discharge.
g
Samples drawn for the central lab should be performed just prior to a scheduled transfusion if applicable. See Section 8.10.
h
Bone marrow assessment for assessment of ineffective erythropoiesis (including myeloid:erythroid ratio) and iron stain. Central assessment for baseline testing of allelic edits as well as
exploratory biomarkers.
i
PRO assessment should be performed as the first assessment at the visit. Subjects who are <18 years old at Screening should continue to complete the EQ-5D-Y and PedsQL, or parent proxy
versions, for the entire study and will not complete the adult PROs when they reach 18 years of age.
j
k
Performance status will be assessed using the Karnofsky Performance Status Scale for subjects ≥16 years old and the Lansky Performance Status Scale for subjects <16 years old. See
Section 8.4.
l
If a subject has not engrafted at M1 visit (defined as ANC ≥500/µL for 3 measurements on 3 consecutive days), the allelic editing assessment should be collected after engraftment and before
the M2 visit.
m
Section 8.7
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8. STUDY ASSESSMENTS
8.1. Demographic/Medical History

Demographics (e.g., sex, age, race, ethnicity), 2 years of transfusion history (include any simple
or exchange transfusions), and 2 years of hospitalization history (including chelation therapy
received) will be recorded. Thalassemia history such as genotype, age of diagnosis, age of first
transfusion and age of initiation of chelation therapy will also be recorded. Past and present
medical history, including targeted medical history and additional medical history considered by
the investigator to be significant will also be recorded.
8.2. Transfusions
Any transfusion received and reason for transfusion (mL packed RBC/kg and number of
transfused units) will be documented from the time of consent through the end of the study.
Additionally, Hb concentration, and weight before transfusion will be documented.
8.3. Vital Signs and Weight/Height

Vital signs (blood pressure [systolic and diastolic], temperature, pulse rate, respiration rate, and
pulse oximetry). The subject will be instructed to rest for at least 5 minutes before vital signs are
assessed. Subject weight (kg) and height (cm) will be measured with shoes off.
8.4. Physical Examination

Physical examinations will be performed by trained medical personnel and should include the
examination of general appearance, head, skin, neck (including thyroid), eyes, ears, nose, throat,
lungs, heart, abdomen (including spleen size), lymph nodes, extremities, vascular and
neurological systems, performance status. Performance status will be assessed using the
Karnofsky Performance Status Scale for subjects ≥16 years old (Appendix 2) and the Lansky
Performance Status Scale for subjects <16 years old (Appendix 3). Breast, anorectal, and genital
examinations will only be performed when medically indicated.
8.5. Electrocardiogram
Subjects should rest for at least 5 minutes before performing the 12-lead electrocardiogram
(ECG). The ECG should be performed with the subject in the supine position. The ECG will be
performed before any other procedures that may affect heart rate, such as blood draws. The ECG
measurement recorded pretreatment on Day 1 will serve as the subject’s baseline for all post-
infusion comparisons. A printout of the ECG traces will be made for safety review by the
investigator and maintained with source documentation. Clinically significant ECG
abnormalities occurring during the study through the Safety Follow-up Visit will be recorded as
AEs.
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8.6. Diffusing Capacity of the Lungs for Carbon Monoxide (DLCO)

DLco (corrected) will be assessed. PFTs will be performed with the subject in a seated position
and should be performed per site’s SOPs for pre-transplant workup and using a calibrated
spirometer.
8.7. Imaging Studies

A transthoracic cardiac echocardiogram (for assessment of LVEF), and cardiac magnetic
resonance imaging (T2* MRI) (for assessment of iron overload) will be performed according to
Table 3 through Table 6 and read by trained medical personnel.
Subjects who develop signs/symptoms of congestive heart failure at any point during the study
are required to have an evaluation will undergo LVEF measurement by echocardiogram.
A R2* MRI of the liver will be performed according to Table 3 through Table 6 for assessment
of iron overload. The scans for the R2* MRI of the liver and cardiac T2* MRI will be reviewed
at a central facility.
Anesthesia for imaging studies may be used per site standard of care and subject age.
Detailed procedures for image acquisition and analysis will be provided in a separate document.
8.8. Adverse Events

All AEs will be assessed, documented, and reported in accordance with ICH GCP Guidelines.
Section 9.1.1 outlines the definitions, collection periods, criteria, and procedures for
documenting, grading, and reporting AEs.
8.9. Laboratory Assessments

Blood and urine samples will be collected according to Table 3 through Table 6 and analyzed as
per Table 7. Additional clinical laboratory tests may be obtained at any time as clinically
necessary for safety purposes at the investigator’s discretion. Some laboratory values (blood for
hemoglobin fractionation, HbF distribution, α-globin mRNA, exploratory and research studies)
should be obtained pre-RBC transfusion, whenever possible.
In certain circumstances, additional testing may be required depending on the subject’s history
and local requirements for cell processing (e.g., malaria, Trypanosoma cruzi).
Females of childbearing potential (as defined in Section 3.6) must have a negative pregnancy test
result at screening, within 3 days prior to starting mobilization, and within 5 days before the
initiation of busulfan conditioning.
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Hematology Serum Chemistry Urinalysis Other blood tests
CBC with differential including: ALT Protein CD34+ cell count

Hemoglobin AST Blood
WBC Bilirubin (total and direct) Nitrite Globin assessment
ANC Albumin Leukocyte esterase (HbF and other Hb
Platelet count Alkaline phosphatase subtypes)
Reticulocyte count Bicarbonate
Nucleated RBCs BUN Allelic editing
MCV (Mean corpuscular volume) Calcium (blood; central
MCHC (Mean corpuscular Chloride laboratory)
hemoglobin concentration) CK Bone Marrow Tests
MCH (Mean corpuscular Creatinine
hemoglobin) Glucose Myeloid:erythroid
RDW (Red blood cell distribution GGTP (Gamma-glutamyl ratio
width)1 transpeptidase)
Magnesium Iron staining
Phosphorous
Potassium Allelic editing (bone
Sodium marrow aspirate;
Total protein central lab) At screening only
Uric acid
Genotyping of HBB,
and alpha loci
(central lab)
Infectious Disease Immunological

Marker Testing2 Testing
HBV (HbcAb, HbsAg, and CD4
NAT) CD8
CD19
HCV (HCAb and NAT) CD16
CD56
HIV-1, HIV-2 IgG
(Antigen/Antibody IgD
immunoassay3; if positive, IgE
then NAT) IgM
IgA
Syphilis4
HTLV-1 Ab5
1
While a subject is on a regular transfusion regimen, samples for central labs should be collected immediately
before transfusion, and must be collected within 7 days before the next scheduled transfusion. After dosing with
CTX001, if the subject is no longer receiving regular transfusions, samples should be collected immediately before a
transfusion and at least 2 weeks after a previous transfusion.
2
Additional infectious disease markers testing may be done if required by local practice (e.g., HTLV-1, HTLV-2,
malaria, tuberculosis, toxoplasmosis, Typanosoma cruzi, or West Nile virus).
3
In line with standard practice for donor screening, reflexive HIV testing is permitted.
4
Subjects must have at least a screening assay for syphilis performed (e.g., rapid plasma reagin [RPR] is preferred).
If positive, a confirmatory test (e.g. FTA-ABS, TPPA) must be done to confirm the presence of active infection.
Local testing algorithms for syphilis may be used, provided they contain these tests.
5
Testing for HTLV-1 Ab may be required depending on subject’s history and characteristics.
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Hemolysis Markers & Iron Studies Coagulation Pregnancy
Erythropoiesis
Haptoglobin Ferritin PT (Prothrombin Serum pregnancy
LDH (Lactate dehydrogenase) Serum iron time) test, if applicable
Soluble transferrin receptor (use the Soluble transferrin aPTT (Activated
local lab if available; central lab receptor partial
may be used) Transferrin thromboplastin time)
Erythropoietin (Epo) (use the local TIBC (Total iron binding INR
lab if available; central lab may be capacity)
used)
8.10. Blood for Biomarker Assessments

Blood samples will be collected according to Table 4 through Table 6 for evaluation of
biomarkers and their ability to characterize the effect of CTX001 and predict treatment
outcomes.
leukocyte DNA.
• Exploratory biomarker analysis of peripheral blood as compared to baseline over time,
including but not limited to expression of α-globin and non-α-globin mRNA in circulating
reticulocytes over time.
quantitation in peripheral blood to assess bulk fetal hemoglobin levels, (2) the proportion of
circulating erythrocytes expressing fetal hemoglobin (F-cells).
8.11. Blood for Exploratory Research Studies and Assay Development

Blood samples will be collected according to Table 4 through Table 6 for potential exploratory
research and assay development to better understand CTX001 and its impact on
hemoglobinopathies.
Samples of apheresed stem cells and CTX001 will be collected during manufacturing for
CTX001 and for exploratory research to better understand CTX001 and its impact on
β-thalassemia. Samples will not impact the final CTX001 dose of subject.
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8.12. Sperm and Oocyte Banking

choose to undergo the procedures (or their legally authorized representative chooses to have
them undergo the procedures) will do so after eligibility is confirmed and prior to busulfan
conditioning (Stage 3A). For pre-pubescent subjects, gonadal tissue collection and
cryopreservation may be performed per local practice and standard of care.
8.13. Biopsies
8.13.1. Liver Biopsy
At screening, liver biopsy should only be performed after all other assessments have been done
to assess eligibility. Subjects will undergo liver biopsy only when it is considered appropriate by
the investigator and according to institutional standards.
8.13.2. Bone Marrow Aspirate

Bone marrow aspirate will be performed once eligibility is confirmed. Samples will be collected
to assess ineffective erythropoiesis and iron content at the site. Bone marrow aspirate samples
will be collected according to Table 4 through Table 6 for central assessment of prospective
allele editing frequency. Additionally, samples will be taken for exploratory research to better
understand CTX001 and hemoglobinopathies and a sample will also be collected for potential
future genome analysis in case of development of malignancy thought to be related to CTX001.
8.14. Patient Reported Outcomes (PROs)

PRO surveys should be performed as the first assessment after obtaining informed consent, as
well as be completed by subjects at the beginning of a study visit prior to any assessments.
8.14.1. EQ-5D-5L and EQ-5D-Y

The EuroQol Questionnaire – 5 dimensions – 5 levels of severity (EQ-5D-5L) questionnaire
assesses an adult subject’s health status in a standardized way and consists of 2 parts: the EQ-5D
descriptive system and the EQ visual analogue scale (VAS).
The EQ-5D-5L descriptive system comprises the same 5 dimensions, mobility, self-care, usual
activities, pain/discomfort, anxiety/depression. Each dimension has 5 levels: no problems, slight
problems, moderate problems, severe problems, and extreme problems. The respondent is asked
to indicate his/her health state by ticking (or placing a cross) in the box against the most
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The EQ VAS records the subject’s self-rated health on a 100-point VAS with endpoints labelled
For subjects under the age of 18, child-friendly versions of EQ-5D are available for
self-completion (EQ-5D-Youth version; EQ-5D-Y) and parent proxy completion (EQ-5D-Y
proxy version) (van Reenen et al., 2014). In this study, the EQ-5D-Y will be administered for
self-completion to subjects 12 to <18 years of age. A parent proxy version is available for
subjects who are unable to complete the questionnaire themselves. Subjects who are <18 years
old at Screening should continue to complete the EQ-5D-Y or parent proxy version for the entire
study; they will not change to the adult version (EQ-5D-5L) when they reach 18 years of age.
The EQ-5D-Y consists of 2 pages: the EQ-5D-Y descriptive system and the EQ VAS. The
descriptive system comprises the same 5 dimensions as the EQ-5D-5L but using child-friendly
wording. Unlike EQ-5D-5L, which utilizes 5 levels, each dimension in the Youth version has 3
levels: no problems, some problems, and a lot of problems. The respondent is asked to indicate
his/her health state by ticking (or placing a cross) in the box against the most appropriate
statement in each of the 5 dimensions. The EQ VAS used in the youth version is identical to the
EQ VAS used in EQ-5D-5L. Similar to EQ-5D-5L, responses can be used to determine a
quantitative measure of health outcome (i.e., health utilities).
8.14.2. FACT-BMT
The Functional Assessment of Cancer Therapy – for adult subjects undergoing bone marrow
physical, social, family, emotional, and functional well-being. The FACT-BMT consists of the
FACT-General (constitutes the core of all subscales) and treatment-specific concerns of bone
marrow transplantation.
Each statement in the FACT-BMT has a five point Likert-type response scale ranging from 0 to
4 (0 = “not at all”; 1 = “a little bit”; 2 = “somewhat”; 3 = “quite a bit”; and 4 = “very much”).
The subject is asked to circle or mark one number per line to indicate his/her response to the
statement as it applies to the past 7 days. Questionnaires are then scored, and the higher the
score, the better the QOL.
This information can be used to provide a holistic assessment that identifies subject’s needs,
which may not be revealed by a standard clinical consultation.
8.14.3. Pediatric Quality of Life Inventory (PedsQL)

As a pediatric version of FACT-BMT does not exist, the Pediatric Quality of Life Inventory
questionnaire (PedsQL - Teen version) will be administered to subjects 12 to <18 years of age
for self-completion. A parent proxy version is available for subjects who are unable to complete
the questionnaire themselves. Subjects who are <18 years old at Screening should continue to
complete the PedsQL or parent proxy version for the entire study and will not complete the
FACT-BMT when they reach 18 years of age.
The PedsQL is a brief, standardized, generic instrument that systematically assesses subjects' and
parents' perceptions of health-related quality of life in pediatric subjects with chronic health
conditions using pediatric cancer as an exemplary model (Varni et al., 2002). The teen version of
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PedsQL (self-report and parent proxy versions) comprises of 23 items across 4 domains
including Physical Functioning, Emotional Functioning, Social Functioning and School
Functioning (Varni et al., 2017). When completing the questionnaire, respondents are asked to
report the degree to which each item has been a problem over the past month by selecting the
most appropriate response on a five-point scale ranging from 0 (never a problem) to 4 (almost
always a problem). Responses are transformed to a 0 to 100 score, with higher scores reflecting
better health-related quality of life.
8.15. Mini-Mental State Examination and Wechsler Abbreviated Scale

of Intelligence
Subjects will be evaluated for cognitive function using the Mini-Mental State Examination
(subjects ≥18 years old at the time of assessment) or Wechsler Abbreviated Scale of Intelligence
(WASI-II) (subjects 12 to <18 years old at the time of assessment) after eligibility is confirmed
and prior to start of mobilization.
9. SAFETY MONITORING AND REPORTING

9.1. Adverse and Serious Adverse Events
9.1.1. Definition of Adverse Events
9.1.1.1. Adverse Event (AE)

An AE is defined as any untoward medical occurrence in a subject during the study; the event
does not necessarily have a causal relationship with the treatment. This includes any newly
occurring event or worsening of a pre-existing condition (e.g., increase in its severity or
frequency) after signing the ICF or assent form (where applicable). A pre-existing condition that
is diagnosed prior to subject signing ICF should be recorded in medical history.
9.1.1.2. Clinically Significant Assessments

Study assessments including laboratory tests, ECGs, Physical Examinations, and vital signs will
be assessed and those deemed to have clinically significant worsening from baseline will be
documented as an AE as shown in Table 8. When possible, a clinical diagnosis for the study
assessment will be provided, rather than the abnormal test result alone (e.g., urinary tract
infection, anemia). In the absence of a diagnosis, the abnormal study assessment itself will be
listed as the AE (e.g., bacteria in urine or decreased hemoglobin) as shown in Table 8.
An abnormal study assessment is considered clinically significant if the subject has 1 or more of
the following:
• Concomitant signs or symptoms related to the abnormal study assessment
• Further diagnostic testing or medical/surgical intervention
• Discontinuation from the study
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Repeat testing to determine whether the result is abnormal, in the absence of any of the above
criteria, does not necessarily meet clinically significant criteria. The determination of whether the
study assessment results are clinically significant will be made by the investigator.
A laboratory value that is Grade 4 will not automatically be a serious adverse event (SAE). A
Grade 4 laboratory value will be an SAE if the subject’s clinical status indicates a life-
threatening AE.
Busulfan will be administered with the intention and expectation of myeloablation. In this
context, Grade 1 and 2 lab abnormalities reflective of and fully explained by busulfan
myeloablation should not be considered clinically significant.
9.1.1.3. Collection of Adverse Events (AEs)

AEs will be monitored and collected as outlined in Table 8.
Table 8 AE and SAE Recording
Study Time Period AEs SAEs

Signing of the ICF to start of All grades of study All SAEs
mobilization procedure-related AEs only
(including AEs possibly related to
transfusion regimen, and excluding
AEs possibly related to fertility
preservation procedures)
Start of first mobilization cycle to All AEs All SAEs
2 weeks after end of last
mobilization cycle
Two weeks after end of last All grades of study All SAEs
mobilization cycle to start of procedure-related AEs only
busulfan conditioning (including AEs possibly related to
transfusion regimen)
Start of busulfan conditioning to All AEs All SAEs
start of CTX001 infusion
Start of CTX001 infusion to Month All AEs All SAEs
24 (End of Study Visit)
1 overall event or diagnosis. AEs for enrolled subjects will be recorded in the CRF and source
document. AEs for subjects who are screened but not subsequently enrolled will be recorded
only in the subject’s source documents. The following data will be documented for each AE:
 Description of the event
 Classification of “serious” or “nonserious”
 Date of first occurrence and date of resolution (if applicable)
 Severity
 Causal relationship to study drug(s)
 Action taken
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 Outcome
 Concomitant medication or other treatment given
study drug(s). Causality will be classified using the categories presented in Table 9.
Table 9: Classifications for AE Causality
Related There is an association between the event and the administration of

investigational study drug, a plausible mechanism for the event to be related
to the investigational study drug and causes other than the investigational
study drug have been ruled out, and/or the event reappeared on re-exposure
to the investigational study drug.
investigational study drug and there is a plausible mechanism for the event
to be related to investigational study drug, but there may also be alternative
etiology, such as characteristics of the subject’s clinical status or underlying
disease.
Unlikely related The event is unlikely to be related to the investigational study drug and
likely to be related to factors other than investigational study drug.
Not related The event is related to an etiology other than the investigational study drug
(the alternative etiology will be documented in the study subject's medical
record).
The investigator will determine and record the severity of all serious and nonserious AEs. The
guidance available at the following website will be consulted: CTCAE version 5.0, Cancer
Therapy Evaluation Program,
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm (Accessed
November 2018). The severity of an AE that does not appear in the CTCAE will be determined
according to the definitions in Table 10:
Table 10: Grading Scale for Adverse Events
Mild Mild level of discomfort and does not interfere with regular activities
(Grade 1)
Moderate Moderate level of discomfort and significantly interferes with regular
(Grade 2) activities
Severe Significant level of discomfort and prevents regular activities
(Grade 3)
Life- Any adverse drug event that places the subject, in the view of the
threatening investigator, at immediate risk of death
(Grade 4)
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Death Any adverse event that results in the death of the subject
(Grade 5)
The outcome will be classified according to the categories shown in Table 11.
Table 11: Classifications for Outcome of an AE
Recovered/Resolved Resolution of an AE with no residual signs or symptoms
Recovered/Resolved Resolution of an AE with residual signs or symptoms

with Sequelae
Not Recovered/Not Either incomplete improvement or no improvement of an AE, such that it

Resolved (Continuing) remains ongoing
Fatal Outcome of an AE is death. “Fatal” will be used when death is at least

possibly related to the AE.
9.1.1.4. Serious Adverse Events (SAEs)

An SAE is any AE that meets any of the following outcomes:
• Fatal (death, regardless of cause, that occurs during participation in the study or occurs
after participation in the study and is suspected of being a delayed toxicity due to
administration of the study drug)
• Life-threatening, such that the subject was at immediate risk of death from the reaction as
it occurred
mobilization/apheresis, central line placement, busulfan conditioning, CTX001 infusion)
do not meet this criterion.
• Persistent or significant disability/incapacity (disability is defined as a substantial
disruption of a person's ability to conduct normal life functions)
• Important medical event that, based upon appropriate medical judgement, may jeopardize
the subject or may require medical or surgical intervention to prevent 1 of the outcomes
listed above (e.g., an allergic bronchospasm requiring intensive treatment in an emergency
room or at home)
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• Engraftment failure: failure to achieve neutrophil engraftment by Day 42 after CTX001

infusion or need to receive backup stem cells at any time during period of neutropenia.
• Development of new malignancy.
associated with an AE (e.g., social hospitalization for purposes of respite care) will not be
considered to indicate an SAE.
“serious,” which is based on subject/event outcome or action described above, and is usually
associated with events that pose a threat to a subject's life or functioning. Seriousness, not
9.1.1.5. Collection of Serious Adverse Events

All SAEs that occur after obtaining informed consent and assent (where applicable) through the
24-Month Visit, regardless of causality, will be reported by the investigator to Vertex Global
Patient Safety (GPS) within 24 hours of identification. In addition, all SAEs that occur after the
24-Month Visit and are considered related to study drug(s) will be reported to Vertex GPS
within 24 hours of identification.
9.1.2. Reporting Serious Adverse Events

SAEs that occur after the 24-Month Visit and are considered related to study drug(s) will be
recorded on the Vertex Organized Safety Information Collection Form (hereafter referred to as
the “SAE Form”) using a recognized medical term or diagnosis that accurately reflects the event.
SAEs will be assessed by the investigator for relationship to the investigational study drug(s) and
possible etiologies. On the SAE Form, relationship to study drug(s) will be assessed only as
related (includes possibly related) or not related (includes unlikely related), and severity
assessment will not be required. For the purposes of study analysis, if the event has not resolved
at the end of the study reporting period, it will be documented as ongoing. For purposes of
regulatory safety monitoring, the investigator is required to follow the event to resolution and
report the outcome to Vertex using the SAE Form.
For SAEs that occur after obtaining informed consent and assent (where applicable) through the
24-Month Visit, the SAE Form will be completed for new/initial events as well as to report
follow-up information on previously reported events. Investigators are asked to report follow-up
information as soon as it becomes available to ensure timely reporting to health authorities.
Please send completed SAE Forms to Vertex GPS via:
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Fax: +1-617-341-6159
In parallel with reporting via the SAE Form described above, the investigator must also
immediately notify the medical monitor of any of the following:
 Malignancies
9.1.3. Expedited Reporting and Investigator Safety Letters

Vertex GPS is responsible for reporting suspected, unexpected, serious adverse reactions
(SUSARs) involving the study drug(s) to all regulatory authorities and participating investigators
in accordance with ICH Guidelines and/or local regulatory requirements, as applicable. In
addition, Vertex GPS or authorized designee, will be responsible for the submission of safety
letters to central ECs.
It is the responsibility of the investigator or designee to promptly notify the local IRB/local EC
of all unexpected serious adverse drug reactions involving risk to human subjects. Investigators
will also be notified of all unexpected, serious, drug-related events (7/15 Day Safety Reports)
that occur during the clinical trial. Each site is responsible for notifying its IRB or EC of these
additional SAEs.
9.2. Pregnancy
Subjects will be counseled to inform the investigator of any pregnancy that occurs during study.
If a subject becomes pregnant during the study, study interventions that may put the fetus at risk
will be stopped immediately.
reported to the medical monitor and Vertex GPS within 24 hours of the site’s knowledge using
the Pregnancy Information Collection Form.
outcome, and the infant will be followed for 1 year after the birth, provided informed consent
and, where applicable, assent is obtained. A separate ICF will be provided to explain these
follow-up- activities. Birth outcomes must be reported to the Vertex GPS using the Pregnancy
Information Collection Form, even if the subject was discontinued from the study.
All reports of congenital abnormalities/birth defects are SAEs. Spontaneous miscarriages should
also be reported and handled as SAEs. Elective abortions without complications should not be
handled as AEs.
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10.3. General Considerations

Continuous variables will be summarized using the following descriptive summary statistics:
the number of subjects (n), mean, SD, median, minimum value (min), and maximum value
(max). The precision of the measurement for each continuous variable will be specified in the
SAP.
Categorical variables will be summarized using counts and percentages. Percentages will be
presented to 1 decimal place.
Uncertainty of estimates will be assessed by confidence intervals. All subject data will be
presented in the subject data listings; listings will display all subjects in the enrolled population,
regardless of whether or not they received study drug. Longitudinal data will be presented by
appropriate time intervals, such as monthly or quarterly depending on the nature of the data.
Baseline value, unless otherwise specified, will be defined as the most recent non-missing
measurement (scheduled or unscheduled) collected during screening and prior to start of
mobilization.
Change (absolute change) from baseline will be calculated as post-baseline value subtracted by
baseline value.
Relative change from baseline will be calculated and expressed in percentage as
100% × (Post-baseline value − Baseline value)/Baseline value.
The treatment-emergent (TE) period will include the time from CXT001 infusion to last study
visit.
Incomplete/missing data will not be imputed, unless specified otherwise. All data will be
evaluated as observed. For subjects who are lost to follow-up or die, analyses will be based on
their available data, before death or loss to follow-up. For subjects who withdraw from the study
but enroll in the long-term follow-up study (Study VX18-CTX001-131), analyses will be based
on their data from both the parent and the long-term follow-up studies up to 2 years after
CTX001 infusion, unless otherwise specified.
Subgroup analyses, such as descriptive summaries of the key efficacy and safety endpoints by
age groups as well as by genotypes, will be provided as appropriate. Details will be described in
the SAP.
10.4. Subject Disposition

Number and percentage of subjects in the following categories will be summarized as
appropriate based on the Enrolled Set:
 Subjects who are mobilized
 Subjects who are dosed with CTX001
 Subjects who are dosed with CTX001 with at least 1 year of follow-up post-CTX001
infusion
 Completed study (24-month post-CTX001 follow-up)
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 Prematurely discontinued the study and the reasons for discontinuation
10.5. Demographic and Baseline Characteristics

The following characteristics will be summarized:
 Age, sex, race, ethnicity, weight, height, genotype
 Targeted medical history
 Results of screening imaging (cardiac, liver, lung) and selected lab tests
 Annualized volume of packed RBC transfusion in 2 years prior to screening
 PRO parameters observed at screening
10.6. Efficacy Analysis

10.6.1. Analysis of Primary Efficacy Endpoint
The response status of the primary efficacy endpoint for a subject is evaluated based on his/her
relative change from baseline in annualized volume of RBC transfusion at least 3 months after
CTX001 infusion and until the time of analysis. Baseline annualized volume of RBC
transfusions will be calculated using records of all transfusion events during the 2 years before
signing of the ICF or the latest rescreening for subjects going through rescreenings. Post-baseline
annualized volume of RBC transfusions will be calculated using transfusions that occur 3 months
after CTX001 infusion until the end of study (Month 24). Transfusion events that occur more
than 3 months after CTX001 infusion and are deemed NOT related to the underlying β-
thalassemia will be adjudicated by an endpoint adjudication committee (EAC). Transfusions not
related to the underlying β-thalassemia, according to EAC, will be excluded from the analysis.
A subject will be considered to have met the primary efficacy endpoint if he/she has achieved
sustained transfusion reduction from baseline by at least 50% at the time of analysis, and if this
reduction has been sustained for a period of at least 6 months.
The proportion of subjects who meet the primary efficacy endpoint will be provided, with a one-
sided P value against a response rate and a two-sided 95% exact Clopper-Pearson CI.
10.6.2. Analysis of Key Secondary Endpoint

The response status of the key secondary efficacy endpoint for a subject is evaluated based on
his/her RBC transfusion at least 3 months after CTX001 infusion and until the time of analysis.
A subject will be considered to have met the key secondary efficacy endpoint if, at the time of
analysis, he/she has achieved sustained transfusion independence, defined as absence of
transfusions for a period of at least 6 months.
The proportion of subjects who meet the key secondary efficacy endpoint will be provided, with
a one-sided P value against a response rate and a two-sided 95% exact Clopper-Pearson CI.
10.6.3. Secondary Efficacy Endpoints

 Proportion of subjects achieving sustained TR12 with the corresponding two-sided 95%
exact Clopper-Pearson CI will be provided. For a subject to be considered as a TR12
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responder, he/she will have to achieve durable transfusion reduction, defined as at least
50% reduction compared to baseline after 3 months post-CTX001 infusion for at least
12 months at the time of analysis. The analysis for sustained TR12 will be similar to the
analysis for sustained TR6.
 Proportion of subjects achieving sustained TI12 with the corresponding two-sided 95%
exact Clopper-Pearson CI will be provided. For a subject to be considered as a TI12
responder, he/she will have to achieve sustained transfusion independence, defined as
absence of transfusions 3 months after CTX001 infusion for at least 12 months at the
time of analysis. The analysis for sustained TI12 will be similar to the analysis for
sustained TI6.
leukocytes will be summarized as a continuous variable over time.
 Proportion of alleles with intended genetic modification present in bone marrow cells will
be summarized as a continuous variable over time.
 HbF levels (pre-transfusion) will be summarized as a continuous variable over time.
 Total hemoglobin concentration (pre-transfusion) will be summarized as a continuous
variable over time.
 Change in PROs will be summarized as a continuous variable over time.
 Change in parameters of iron overload, including in LIC and CIC from baseline as
assessed by R2* MRI for LIC and T2* MRI for CIC, and change in serum ferritin level
from baseline over time
o Change in serum ferritin level as compared with baseline will be summarized as a
o Change in Liver R2* at Months 12 and 24 compared with baseline will be
o Change in Cardiac T2* at Months 12 and 24 compared with baseline will be
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10.7. Safety Analysis

Safety analyses will include:
• Neutrophil engraftment and platelet engraftment
• Frequency and severity of AEs from signing of informed consent through the Month 24
Visit, as specified in Table 8, and as assessed by CTCAE v5.0.
• Incidence of TRM at 100 days and 1 year post CTX001 infusion. TRM defined as death
• Laboratory results, ECGs, and vital signs
For all-cause mortality Kaplan-Meier method will be used. The number and percentage of
subjects with TRM will be summarized at 100 days and 1 year post CTX001 infusion, where
TRM is defined as death at least possibly related to the transplantation procedure as assessed by
the investigator. Relatedness between SAEs leading to death and transplantation will be as
assessed by the investigators. If an SAE is assessed as being at least possibly related to the
transplantation procedure, the death will be classified as transplant-related.
10.7.1. Engraftment
Neutrophil engraftment is defined as the first day of 3 measurements of absolute neutrophil count
(ANC) ≥500/µL on 3 consecutive days, achieved within 42 days post CTX001 infusion, without
use of the unmodified CD34+ cells after reaching the nadir, defined as ANC < 500/uL.
Platelet engraftment is defined as the first day of 3 consecutive measurements of platelet
≥20,000/μL on 3 different days after CTX001 infusion, without a platelet transfusion in the past
7 days, after reaching the nadir, defined as platelet < 20,000/uL.
The number and percentage of subjects achieving successful engraftment, together with the 95%
confidence interval will be provided.
Time to neutrophil engraftment will be analyzed by the Kaplan-Meier method. Engraftment
failure is defined as not achieving neutrophil engraftment by Day 42 after CTX001 infusion, or
receipt of backup stem cells. The number and percentage of subjects with neutrophil engraftment
failure will be summarized.
Time to platelet engraftment will be analyzed by the Kaplan-Meier method.
10.7.2. Adverse Events

AEs will be coded according to Medical Dictionary for Regulatory Activities (MedDRA) and
summarized by AE recording period specified in Table 8.
Details for imputing missing or partial start dates of AEs are described in the SAP.
 AEs by strongest relationship
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 AEs by maximum severity

 Serious AEs
 AEs leading to death
Summaries will be presented by Medical Dictionary for Regulatory Activities (MedDRA)
System Organ Class and Preferred Term using frequency counts and percentages (i.e., number
and percentage of subjects with an event). When summarizing the number and percentage of
subjects with an event, subjects with multiple occurrences of the same AE or a continuing AE
will be counted once. Only the maximum severity level will be presented in the severity
summaries, and the strongest relationship level will be presented in the relationship summaries.
10.7.3. Clinical Laboratory Assessments

hematology and chemistry results, including coagulation studies will be summarized in
International System of Units (SI) by visit. Laboratory values will be analyzed based on specific
AE period.
10.7.4. Electrocardiogram
will be provided visit for the following standard 12 lead ECG measurements: RR (msec), HR
HR intervals (QTcF [msec]). ECGs will be summarized per the AE period.
10.7.5. Vital Signs

10.7.6. Physical Examination

PE findings will be presented as an individual subject data listing only.
10.8. Exploratory Analysis

Descriptive statistics will be used to describe the following parameters (unless stated otherwise).
 Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells)
from baseline (pre-transfusion) at Months 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, 24
 Expression of α-globin and non-α-globin mRNA in circulating reticulocytes at baseline,
Months 3, 6, 9, 12, 18, 24
 Assessment of erythropoiesis on bone marrow analysis compared with baseline over time
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 Correlations of response markers (transfusions, total and fetal hemoglobin, with pre-
treatment variables (e.g., α/non-α globin ratio, subject genotype), cell dose and % edited
cells in final product
clinical study report.
10.9. Interim Analyses
10.10. Procedures for Handling Missing Data

In general, no missing data will be imputed, unless specified otherwise. All data will be
evaluated as observed. For subjects who are lost to follow-up or die, analyses will be based on
their available data, before death or loss to follow-up. For subjects who withdraw from the study
but enroll in the long-term follow-up study, analyses will be based on their data from both the
parent and the long-term follow-up studies up to 2 years after CTX001 infusion, unless otherwise
specified.
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10.11. Steering Committee

The Steering Committee (SC) will provide expert guidance on the study execution strategy and
help with increasing study awareness and enrollment. Details of the SC structure and function,
frequency of meetings, and data planned for review will be included in the SC charter.
10.12. Endpoint Adjudication Committee (EAC)

group of experts with appropriate clinical scientific background to evaluate packed RBC
transfusion needs. The EAC will be responsible for distinguishing RBC transfusions related to
the underlying disease of β-thalassemia and transfusions administered for the purpose of acute,
clinical needs (i.e. non-disease related) post-CTX001 infusion.
Details of the EAC structure, function, and frequency of meetings, will be included in the EAC
charter, which will be finalized prior to the first meeting.
11. DATA COLLECTION AND MANAGEMENT

This study is open-label. Subjects, the study sponsor, and investigators will be aware that each
subject is planned to be treated with CTX001. Data handling procedures designed to maintain the
study credibility and validity in this open-label single arm study are described in a separate
document.
11.1. Monitoring
Before an investigational site can enter a subject into the study, a representative of the Sponsor
or designee will visit the investigational study site to:
 Determine the adequacy of the facilities
 Discuss with the investigator(s) and other personnel their responsibilities with regard to
protocol adherence, and the responsibilities of the Sponsor or designee. This will be
documented in a Clinical Study Agreement between the Sponsor and the investigator.
Monitoring and auditing procedures developed or approved by the Sponsor will be followed to
comply with Good Clinical Practice (GCP) guidelines. On-site checking of the CRFs/SAE Forms
for completeness and clarity, cross-checking with source documents, and clarification of
administrative matters will be performed.
The study will be monitored by the Sponsor or designee. Monitoring will be done by personal
visits from a representative of the Sponsor or designee (study site monitor), who will review the
CRFs/SAE Forms and source documents. The study site monitor will ensure that the
investigation is conducted according to the protocol design and regulatory requirements.
Additionally, during the conduct of the study, a study site monitor will have regular contact with
investigational site, for the following reasons:
 Provide information and support to the investigator(s)
 Confirm that facilities remain acceptable
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 Confirm that the investigational team is adhering to the protocol, that data is being
accurately recorded in the CRFs, and that investigational product accountability checks
are being performed
The monitor will be available between visits if the investigator(s) or other staff needs
information or support.
11.2. Electronic Data Capture (EDC)

The Sponsor will provide the study sites with secure access to and training on the EDC
application sufficient to permit study site personnel to enter or correct information in the CRFs
on the subjects for which they are responsible.
A CRF will be completed for each consented study subject. It is the investigator's responsibility
to ensure the accuracy, completeness, clarity, and timeliness of the data reported in the subject's
CRF. Source documentation supporting the CRF data will indicate the subject's participation in
and subject status.
The audit trail entry will show the user's identification information and the date and time of any
including any changes made to the CRFs, to endorse the final submitted data for the subjects for
The Sponsor will retain the CRF data and corresponding audit trails. A copy of the final archival
CRF in the form of a CD or other electronic media will be placed in the investigator's study file.
11.3. Audits and Inspections

Authorized representatives of the sponsor, a regulatory authority, an EC or an IRB may visit the
site to perform audits or inspections, including source data verification. The purpose of a sponsor
audit or inspection is to systematically and independently examine all study-related activities and
documents to determine whether these activities were conducted, and data were recorded,
analyzed, and accurately reported per the protocol, GCP guidelines of the ICH, and any
applicable regulatory requirements. The investigator should contact the sponsor immediately if
contacted by a regulatory agency about an inspection.
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12. QUALITY CONTROL AND QUALITY ASSURANCE

To ensure compliance with GCPs and all applicable regulatory requirements, the sponsor may
conduct a quality assurance audit. Please see Section 11.3 for more details regarding the audit
process.
13. Ethical And Regulatory Considerations
13.1. Ethics Committee (EC) / Institutional Review Board (IRB)

13.2. Ethics Review

subjects.
13.3. Conduct of the Study

The study will be performed in accordance with ethical principles that have their origin in the
Declaration of Helsinki and are consistent with ICH/GCP and applicable regulatory requirements
13.4. Written Informed Consent

The PI(s) at each center will ensure that the subject is given full and adequate oral and written
information about the nature, purpose, possible risk and benefit of the study. Subjects must also
be notified that they are free to discontinue from the study at any time. The subject should be
given the opportunity to ask questions and allowed time to consider the information provided.
A signed and dated ICF must be obtained from the subject or a legal representative or guardian
(if applicable), and assent will be obtained from the subject (if applicable), before conducting
any study procedures.
The PI(s) must maintain the original, signed ICF. A copy of the signed ICF must be given to the
subject.
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Whenever important new information becomes available that may be relevant to the subject’s
consent, the written ICF and any other written information provided to subjects will be revised
by the Sponsor or designee and be submitted again to the IRB/EC for review and favorable
opinion. The agreed upon, revised information will be provided to each subject in the study for
signing and dating. The investigator will explain the changes to the previous version.
14. DATA HANDLING AND RECORDKEEPING
14.1. Inspection of Records

The Sponsor or designee will be allowed to conduct site visits to the investigation facilities for
monitoring any aspect of the study. The investigator agrees to allow the monitor to inspect the
product storage area, hospital pharmacy product accountability records, subject charts and study
source documents, and other records relative to study conduct.
14.2. Retention of Records

The PI must maintain all documentation relating to the study for a period of 2 years after the last
marketing application approval, or if not approved 2 years following the discontinuance of the
test article for investigation. If it becomes necessary for the Sponsor or the Regulatory Authority
to review any documentation relating to the study, the investigator must permit access to such
records. If maintaining this documentation is no longer possible at the site, the investigator must
notify the Sponsor.
14.3. Subject Privacy and Confidentiality

laws and regulations, all data provided to the Sponsor or designee, study reports, and
communications relating to the study will identify subjects by assigned subject numbers and
access to subject names linked to such numbers shall be limited to the site and the study
physician and shall not be disclosed to the Sponsor or designee. As required by applicable laws
and regulations in the countries in which the study is being conducted, the investigator will allow
the Sponsor and/or its representatives access to all pertinent medical records to allow for the
verification of data gathered and the review of the data collection process. The regulatory
authorities in other jurisdictions, including the EC/IRB, may also request access to all study
records, including source documentation, for inspection.
15. PUBLICATIONS AND CLINICAL STUDY REPORT

15.1. Publication of Study Results
Sponsor. The investigator will hold such information in confidence and will not disclose the
information to any third party except to such of the investigator's employees and staff as have
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whom disclosure is necessary to evaluate that information. The investigator will not use such
if the parties decide to proceed with the study, for the purpose of conducting the study.
partners and associates, health authorities in different countries, the FDA, and other government
agencies. The investigator also understands that, to allow for the use of the information derived
from the clinical study, the investigator has the obligation to provide the Sponsor with complete
test results and all data developed in the study.
conditions of written agreement between the Sponsor and the investigator and/or the
investigator's institution.
15.2. Clinical Study Report

After completion of the study, a clinical study report, written by the Sponsor or designee in
accordance with the ICH E3 Guideline, will be submitted in accordance with local regulations.
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17. APPENDICES
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APPENDIX 1. BUSULFAN PK COLLECTION

Recommendations for dosing Q6H: Samples for busulfan PK analysis should be collected at the
above.
alternatively, use a busulfan test dose prior to myeloablation to pre-determine the dose, with
adjustments will be included in the eCRF.
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APPENDIX 2. KARNOFSKY AND LANKSY PERFORMANCE STATUS

SCALES
Source: Center for International Blood and Bone Marrow Research. 2009.
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Page 1 of 80
1 TITLE PAGE
Clinical Study Protocol

A Phase 1/2 Study to Evaluate the Safety and
Efficacy of a Single Dose of Autologous
CRISPR-Cas9 Modified CD34+ Human Hematopoietic
Stem and Progenitor Cells (CTX001) in Subjects With
Severe Sickle Cell Disease
Study Number: CTX001-121
EudraCT Number: 2018-001320-19
Date of Protocol: 13 April 2018
Baarerstrasse 14
CH 6300 Zug, Switzerland
CONFIDENTIAL
This document contains confidential information. Any use, distribution, or disclosure without the prior written consent
of CRISPR Therapeutics AG is strictly prohibited except to the extent required under applicable laws or regulations.
Persons to whom the information is disclosed must be informed that the information is confidential and may not be
further disclosed by them.
Protocol CTX001-121, Version 1.0 Pg. 1

Protocol CTX001-121, Version 1.0 Page 2 of 80
2 PROTOCOL SYNOPSIS
Title A Phase 1/2 Study to Evaluate the Safety and Efficacy of a Single Dose of
Autologous CRISPR-Cas9 Modified CD34+ Human Hematopoietic Stem and
Progenitor Cells (CTX001) in Subjects With Severe Sickle Cell Disease
Brief Title A Study to Evaluate the Safety and Efficacy of a Single Dose of CTX001 in
Subjects With Severe Sickle Cell Disease
Clinical Phase and Phase 1/2 Safety and Efficacy Study

Clinical Study Type
Objectives Primary Objective

Evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
modified CD34+ human hematopoietic stem and progenitor cells (hHSPCs)
(CTX001) in subjects with severe sickle cell disease (SCD)
Secondary Objectives
• Assess the effects of infusion of CTX001 on disease-specific events and
clinical status
• Quantify gene editing efficiency
Exploratory Objective
Assess the ability of biomarkers to characterize CTX001 effect and predict
treatment outcomes
Endpoints Primary Endpoints

Safety Endpoints
• Successful neutrophil engraftment within 42 days after CTX001 infusion
• Time to neutrophil engraftment
• Time to platelet engraftment
• Safety and tolerability assessments based on adverse events (AEs), clinical
laboratory values, and vital signs
• Transplant-related mortality (TRM) within 100 days after CTX001 infusion
• TRM within 1 year after CTX001 infusion
• Proportion of subjects with sustained fetal hemoglobin (HbF) ≥20% for at
least 3 months starting 6 months after CTX001 infusion, in the absence of
treatment with hydroxyurea (HU)
• Relative change from baseline in annualized rate of severe vaso-occlusive
crises (VOC), starting 6 months after CTX001 infusion
Secondary Endpoints
• Reduction in annualized rate of severe VOC from baseline by at least 50%,
starting 6 months after CTX001 infusion
• Reduction in annualized rate of severe VOC from baseline by at least 65%,
CRISPR Therapeutics AG Confidential Information

• Absence of severe VOC events for at least 12 months at the time of the
analysis
• Change from baseline in annualized duration of hospitalization for severe
VOC, starting 6 months after CTX001 infusion
• Proportion of subjects with sustained HbF ≥20% for at least 3 months,
starting 3 months after CTX001 infusion, in the absence of treatment with HU
• Proportion of subjects with sustained HbF ≥20% for at least 3 months,
starting at the time of CTX001 infusion, in the absence of treatment with HU
• Change in number of units of red blood cells (RBC) transfused for
SCD-related indications over time
• HbF concentrations over time
• Hemoglobin (Hb) concentrations over time
• Proportion of alleles with intended genetic modification present in peripheral
blood leukocytes over time
• Proportion of alleles with intended genetic modification present in bone
marrow cells over time
• Change in patient reported outcomes (PROs) over time using weekly
Pain-scale (11 point numerical rating scale [NRS]), EuroQol Quality of Life
Scale (EQ-5D-5L), functional assessment of cancer therapy-bone marrow
transplant (FACT-BMT), Patient-reported Outcome Measurement
Information System (PROMIS)-Fatigue, PROMIS-Cognitive function, and
Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me)
• Change in hemolytic index as measured by principal component analysis of
4 markers of hemolysis (reticulocyte count, serum concentrations of aspartate
transaminase, lactate dehydrogenase [LDH], and total bilirubin) over time
• Change in tricuspid regurgitant jet velocity (TRV) over time
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin
(F-cells) over time
• Change in inflammatory and endothelial activation markers over time
Number of Subjects Up to 12 subjects with possible expansion to a total of up to 45 subjects
Study Population Subjects 18 to 35 years of age (inclusive at time of informed consent) with
documented βS/βS genotype who have severe SCD. Severe SCD is defined by the
occurrence of at least 2 of the following events each year during the 2-year period
before screening, while receiving appropriate supportive care (e.g. pain
management plan, HU if indicated) as judged by the investigator:
• Acute pain event that requires a visit to a medical facility and administration
of pain medications (opioids or intravenous [IV] non-steroidal
anti-inflammatory drugs [NSAIDs]) or RBC transfusions
• Acute chest syndrome, as indicated by the presence of a new pulmonary
infiltrate associated with pneumonia-like symptoms, pain, or fever
• Priapism lasting >2 hours
• Splenic sequestration
Investigational Drug Drug product: CTX001

Description: CTX001 is composed of autologous CD34+ hHSPCs modified with

CRISPR-Cas9 at the erythroid lineage-specific enhancer of the BCL11A gene.

Route of administration: IV infusion
Study Design This is a single-arm, open-label, multi-site, single-dose, Phase 1/2 study in
subjects with severe SCD. The study will evaluate the safety and efficacy of a
single dose of autologous CRISPR-Cas9 modified hHSPCs (CTX001) and will
include up to 12 subjects, 18 to 35 years of age, inclusive. The study may be
expanded to include a total of up to 45 subjects.
Stage 1: Screening and Pre-mobilization Period
During this period, subjects who meet the inclusion criteria have the option to
undergo fertility preservation via cryopreservation of oocyte or sperm. After
eligibility is confirmed, subjects will begin RBC transfusions (simple or
exchange) 8 (±2) weeks before the planned start of mobilization and will continue
receiving these transfusions until they begin busulfan conditioning. The goal of
these RBC transfusions is to target hemoglobin S (HbS) level of <30% of total Hb
while keeping total Hb concentration ≤11 g/dL. Treatment with HU should be
stopped at least 6 weeks before planned mobilization.
Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection, CTX001
Manufacture and Disposition
Each subject will undergo stem cell mobilization with plerixafor only. Peripheral
blood mononuclear cells (PBMC) will be collected by apheresis.
On Day 1, subjects will receive plerixafor .
Subjects will undergo apheresis for 2 or 3 consecutive days to collect CD34
hHSPC. The targeted CD34+ cell collection is at least 15 × 106 CD34+ cells/kg for
manufacturing of CTX001 in order to achieve a minimum target dose of 3 × 106
CD34+ cells/kg. An additional 2 × 106 CD34+ cells/kg will be collected as backup
for rescue therapy in an event of non-engraftment with CTX001.
If the first mobilization and apheresis cycle does not yield enough cells for both
the minimum CTX001 product and safety backup or if a subject cannot complete
apheresis, up to 2 additional mobilization and apheresis cycles will be allowed to
collect additional cells. The additional mobilization cycle will be initiated at least
14 days after the first day of the prior mobilization cycle and no more than
60 days after the end of the prior cycle.
Stage 3: Myeloablative Conditioning (Stage 3A) and Infusion of CTX001 (Day 1,
Stage 3B)
Stage 3A - Myeloablative Conditioning: During CTX001 manufacturing and
before the planned start of busulfan conditioning, subjects will continue to receive
simple or exchange RBC transfusions with the goal to maintain HbS level of
<30% of total Hb while keeping total Hb concentration ≤11 g/dL.
If the planned start of busulfan conditioning is >4 months after completion of
mobilization, the investigator may stop the RBC transfusion regimen and restart
HU for those subjects who have been previously treated with HU. If RBC
transfusion regimen is interrupted, subjects should begin RBC transfusions
(simple or exchange) 8 (±2) weeks before the planned start of busulfan
conditioning with the goal to maintain HbS level of <30% of total Hb while
keeping total Hb concentration ≤11 g/dL. If HbS level is >30% of total Hb within
7 (±3) days before the planned start of busulfan conditioning, subjects will receive
1 exchange transfusion with the goal to ensure HbS level is <30% before start of
busulfan conditioning.
After the CTX001 product is received at the site and it has been confirmed that the

the subjects, safeguarding their interests, and ensuring the quality and integrity of
the trial. The DMC will conduct reviews of study data as outlined in the DMC
charter. The DMC may recommend that the Sponsor suspend enrollment, amend
the study, or discontinue the study at any time.
The DMC will review available engraftment and safety data for the first subject
before the second subject will undergo myeloablation. The DMC will also review
data from the second subject if the second subject infused with CTX001
experiences Grade ≥3 AEs other than those typically associated with busulfan
conditioning or autologous transplant procedure, before the remaining subjects
can undergo myeloablation and CTX001 infusion.
Further, the DMC will review safety and efficacy data after at least subjects
have received CTX001 infusion in order to recommend whether or not to expand
the study (expansion cohort).

3 SCHEDULE OF ASSESSMENTS
Schedules of assessments are in Table 3-1, Table 3-2, Table 3-3, Table 3-4 and Table 3-5.
Table 3-1 Study CTX001-121: Screening (Stage 1)

Screening
Event/Assessment (Up to 8 Weeks) Comments
Informed consent X
PRO assessment (Pain-scale X Perform as the first assessment after
[11 point NRS], ASCQ-Me, signing informed consent (Section 11.8)
PROMIS-Fatigue,
PROMIS-Cognitive function,
FACT-BMT, and EQ-5D-5L)
Demographics X Section 11.1
Medical history X Including medical records on SCD-related
complications and associated treatment
including medical facility visits, in-patient
hospitalizations and transfusion records
for at least 2 years before consent will be
collected
Full physical examination X Including assessment of spleen size
(Section 11.10.3)
Performance status X Section 11.10.3
Height and weight X Measure with shoes off
Vital signs X Includes blood pressure (systolic and
diastolic), temperature, pulse rate,
respiration rate, and pulse oximetry
(Section 11.10.3)
SCD genotyping (central X All subjects will be genotyped for SCD
laboratory) (alpha and HBB loci) to confirm βS/βS
genotype. Results will be required before
busulfan conditioning begins.
Serum chemistry X Section 11.10.2
Coagulation X Section 11.10.2
Hematology X Section 11.10.2
Serum pregnancy test X For females of childbearing potential
Hemoglobin fractionation X HbF for establishing eligibility; will also
(central laboratory) include HbS, HbA2, HbA, and other
assessments.
Infectious disease marker testing X Section 11.10.2

Urinalysis X Section 11.10.2
12-lead ECG X Section 11.10.5
Brain MRI/MRA X To confirm absence of active Moyamoya
disease. If a brain MRI/MRA has been
performed within 3 months before date of
consent, these results may be used, unless
repetition of the assessment is clinically
indicated (Section 11.4)
Echocardiogram X LVEF and TRV assessment (Section 11.4)
DLco [corrected] X Section 11.3
Adverse event assessment X Continuous from ICF signing


Screening
Prior and concomitant X Continuous from ICF signing
medications

Protocol CTX001-121, Version 1.0
Table 3-2 Study CTX001-121: Pre-mobilization Period and Autologous CD34+ Stem Cell Apheresis (Stage 1 and Stage 2)
Mobilization and Apheresis
After
Eligibility
Confirmation
(Before Day -5
Mobilization to Day 3
Event/Assessment Start) a Day -1a Day 1 Day 2 (if needed) Comments
Allelic editing (central X Peripheral blood sample; Before scheduled transfusion (if applicable)
laboratory) (Section 11.5)
Immunological testing X Before scheduled transfusion (if applicable) (Section 11.10.2)
HbF distribution, F-cells X Before scheduled transfusion (if applicable) (Section 11.5)
(central laboratory)
Inflammatory and endothelial X Before scheduled transfusion (if applicable) (Section 11.5)
activation markers (central
laboratory)
Bone marrow aspirate (central X Baseline sample for genomic analysis, allelic editing, and exploratory
laboratory) biomarkers (Section 11.6)
Exploratory biomarker samples X Section 11.5.1
Sperm or oocyte banking X (optional) If requested by subject; prior to busulfan conditioning (Section 11.7)
Infectious disease marker X Should be repeated within 10 days before the planned apheresis day
testing (Section 11.10.2)
Mini-Mental State Examination X Section 11.9
Hemolysis markers X Section 11.10.2
Females of childbearing potential only. Must be performed within 3 days of start
Serum pregnancy test X
of mobilization
Eligibility for Per site’s standard guidelines. Subject must be hemodynamically stable and have
mobilization/apheresis X no active infection per investigator judgment
re-confirmed
Abbreviated physical
X X X X Before start of apheresis on Days 1 to 3 (Section 11.10.3)
examination
Performance status X X X X Before start of apheresis on Days 1 to 3 (Section 11.10.3)
Before start of apheresis on Days 1 to 3; Includes blood pressure (systolic and
Vital signs X X X X diastolic), temperature, pulse rate, respiration rate, and pulse oximetry
(Section 11.10.3)
Weight X With shoes off
12-lead ECG X Before start of apheresis or any blood draws (Section 11.10.5)
Hematology X X X X Collect sample before start of apheresis (Section 11.10.2)

Pg. 9
Protocol CTX001-121, Version 1.0 Page 10 of
80
After
Eligibility
Confirmation
(Before Day -5
Mobilization to Day 3
Event/Assessment Start) a Day -1a Day 1 Day 2 (if needed) Comments
Serum chemistry X X X X Collect sample before start of apheresis (Section 11.10.2)
Coagulation X X X X Collect sample before start of apheresis (Section 11.10.2)
Placement of central venous X (if needed)
catheter for apheresis
Pre-mobilization transfusion Refer to Table 3-5
X
regimen
.
X
Plerixafor administration X X Plerixafor will be administered on Day 3 only if apheresis collection is planned
(if needed)
for Day 3.
Day 1: before plerixafor administration and before start of apheresis
CD34+ count X X X Days 2 to 3: before plerixafor administration and before start of apheresis
Sites may perform additional CD34+ count per local standards
Apheresis and shipment of Cells should be shipped to central manufacturer at the end of the first and second
CD34+ stem cells to central X X collection day. For subsequent cycles after the first mobilization/apheresis cycle,
manufacturer Days 2 and 3 of apheresis will be performed as needed.
On Day 2, CD34+ cells will be collected for backup only if sufficient CD34+
Apheresis for CD34+ backup
X X cells are collected for manufacturing. Cells collected for backup dose will be
stem cells
stored at the study site.
Adverse event assessment Continuous from ICF signing
Prior and concomitant Continuous from ICF signing
medications
a
Pg. 10

Table 3-3 Study CTX001-121: Myeloablative Conditioning and Infusion of CTX001 (Stages 3A/3B)
Pre-conditioning Conditioning Infusion

30 (± 2) Days 3 to 7 Days Before Day -5 to -2 Day 1
Before Busulfan Busulfan Initiationa
Event/Assessment Initiationa Comments
Serum pregnancy test X Females of childbearing potential only. Preferably within 5 days of
starting busulfan
Abbreviated physical X X X Section 11.10.3
examination
Performance status X X X Section 11.10.3
Vital signs X X X Includes blood pressure (systolic and diastolic), temperature, pulse
rate, respiration rate, and pulse oximetry.
Day 1: before infusion and every 30 minutes after infusion up to 2
hours post-infusion (Section 11.10.3)
12-lead ECG X X Day 1: ECGs before start of infusion (Section 11.10.5)
Hematology X X X Day 1: Collect laboratory assessments before start of infusion
(Section 11.10.2)
Serum chemistry X X X Day 1: Collect laboratory assessments before start of infusion
(Section 11.10.2)
Eligibility for myeloablation X X 30 (± 2) days before busulfan initiation: If screening and
reconfirmed pre-conditioning visits are ≥4 months apart, repeat pulmonary
function tests and echocardiograph
3 to 7 days before busulfan initiation: Review most recent
assessments (within 7 days prior to conditioning) as per site’s
standard guidelines for autologous stem cell transplant.
Pre-conditioning transfusion X Refer to Table 3-5. Treatment with HU should be stopped at least
regimen 6 weeks before planned mobilization.
Confirm rescue cells are X
cryopreserved in acceptable
condition at the site
Confirm CTX001 has been X
received in acceptable condition
at the site prior to initiating
busulfan
Test dose of busulfan to X (optional)
determine optimal busulfan dose
Anti-seizure prophylaxis X Initiate anti-seizure prophylaxis at least 12 hours before first dose
of busulfan per hospital guidelines
IV busulfan administration X Section 9.11.3
Busulfan PK X Blood sample will be collected for PK analysis for busulfan. If PK
analysis is not available locally for daily results, perform analysis
on first and third day of busulfan dosing as per Appendix 16.1.
Pg. 11

Pre-conditioning Conditioning Infusion

30 (± 2) Days 3 to 7 Days Before Day -5 to -2 Day 1
Before Busulfan Busulfan Initiationa
Event/Assessment Initiationa Comments
IV infusion of CTX001 X Subjects should be pre-medicated with an antihistamine
(diphenhydramine hydrochloride) and an antipyretic
(acetaminophen or paracetamol) per institutional guidelines
(Section 10.1.4)
Adverse event assessment Continuous from ICF Signing
Prior and concomitant Continuous from ICF Signing
medications
a
Assessments may be performed over multiple days of visits.
Pg. 12

Table 3-4 Study CTX001-121: Post-infusion Through Follow-up (Stage 4)
Daily Follow-upa
From D+30 b
D+60 D+90 D+120 D+150 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 ETFc
Event/ M1 M2 M3 M4 M5 M6 M9 M12 M15 M18 M21 M24
Until
(±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d)
Assessment Discharge Comments
Vital signs X X X X X X X X X X X X X X Includes blood pressure (systolic
and diastolic), temperature,
pulse rate, respiration rate, and
pulse oximetry.
(Section 11.10.3)
Abbreviated X X X X X X X X X X X X X X Section 11.10.3
physical
examination
Performance status X X X X X X X X X X X X X X Section 11.10.3
Post CTX001 X Refer to Table 3-5
infusion transfusion
regimen
Hematology X X X X X X X X X X X X X X Section 11.10.2
Serum chemistry X X X X X X X X X X X X X X Section 11.10.2
Coagulation X X X X X X X X X X X X X X Weekly until discharge
(Section 11.10.2)
Hemolysis markers X X X X X X X X X X Section 11.10.2
Immunological X X X X X Section 11.10.2
testing
Allelic editing X X X X X X X X X X X Before scheduled transfusion (if
(central laboratory) applicable) (Section 11.5)
Hemoglobin X X X X X X X X X X X X X Before scheduled transfusion (if
fractionation applicable) (Section 11.5)
HbF distribution, X X X X X X X X X X X X X Before scheduled transfusion (if
F-cells (central applicable) (Section 11.5)
laboratory)
Inflammatory and X X X X X X Before scheduled transfusion (if
endothelial applicable)
activation markers (Section 11.5)
Exploratory X X X X X X Before scheduled transfusion (if
biomarker blood applicable)
samples (central (Section 11.5.1)
laboratory)
Bone marrow X X X X For assessment of allelic editing,
aspirate (central and exploratory biomarkers
DLco [corrected] X X X Section 11.3
Pg. 13

Daily Follow-upa
From D+30 b
D+60 D+90 D+120 D+150 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 ETFc
Event/ M1 M2 M3 M4 M5 M6 M9 M12 M15 M18 M21 M24
Until
(±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d)
Assessment Discharge Comments
Echocardiograph X X X X X TRV assessment (Section 11.4)
12-lead ECG X X X Section 11.10.5

PRO assessments X X X X X X Assessment should be
(EQ-5D-5L, performed as the first
FACT-BMT, assessment at any visit if
PROMIS-Cognitive possible (Section 11.8)
and ASCQ-Me)
PRO assessments X X X X X X X X X X X PRO assessments should be
(Pain-Scale performed weekly starting at
[11 point NRS], M3; Assessment should be
PROMIS-Fatigue) performed as the first
assessment at any visit if
possible (Section 11.8)
Adverse event Continuous from ICF signing
collection
Prior and Continuous from ICF signing
concomitant
medication
a
b
Month (M) is defined as 30 days. If Day 30 occurs while the subject is still hospitalized, the daily assessments required prior to discharge should be done in addition to the Day 30 assessments.
c
Early Termination of Follow-Up (ETF). Assessments performed within 2 weeks of ETF visit should not be repeated. Echocardiograph, DLco, and bone marrow aspirate should not be repeated if
performed within 6 months of ETF visit.
Pg. 14

Table 3-5 Study CTX001-121: Transfusion Regimen

Pre-mobilization During Pre-conditioning After Start of
Post CTX001
Event/ Mobilization Busulfan to CTX001 Notes
Infusion
Assessment 8 (±2) weeks and Apheresis 8 (±2) weeks 7 (±3) days Infusion
a
All transfusions of RBC products should be
depleted of leukocytes and matched for at least the
ABO blood group, the Kell (K) blood group, and
Rh antigens C, D, and E
b
Initiate RBC transfusion (simple or exchange)
8 (±2) weeks before planned start of mobilization
with a goal to target HbS level of <30% of total Hb
while keeping total Hb concentration ≤11 g/dL
c
Continue or reinitiate RBC transfusion (simple or
RBC transfusiona
exchange) 8 (±2) weeks before start of busulfan
(simple or Xb None Xc Xd None Xe
conditioning with a goal to target HbS level
exchange)
of <30% of total Hb while keeping total Hb
concentration ≤11 g/dL
d
If HbS level is >30% of total Hb subjects will
receive 1 exchange RBC transfusion with a goal to
ensure HbS level of <30% of total Hb while
keeping total Hb concentration ≤11 g/dL before
start of conditioning.
e
As needed to maintain Hb ≥7 g/dL per
institutional standards.
Platelet
X Per institutional standards
transfusions
HbS and Hb
X X X
levels
Pg. 15

4 TABLE OF CONTENTS
1 Title Page ................................................................................................................................ 1

2 Protocol Synopsis ................................................................................................................... 2
3 Schedule of Assessments........................................................................................................ 7
4 Table of Contents ................................................................................................................. 16
List of Tables............................................................................................................................ 19
List of Figures .......................................................................................................................... 20
List of Abbreviations................................................................................................................ 21
Glossary of Terms .................................................................................................................... 24
5 Introduction.......................................................................................................................... 25
5.1 Background..................................................................................................................... 25
5.1.1 Hemoglobinopathies ............................................................................................... 25
5.1.2 Sickle Cell Disease ................................................................................................. 25
5.2 Therapeutic Rationale ..................................................................................................... 26
5.2.1 CRISPR-Cas9 Gene Editing Approach .................................................................. 27
5.2.2 BCL11A Erythroid-lineage Specific Enhancer for Editing..................................... 29
5.2.3 CTX001 .................................................................................................................. 30
5.3 Nonclinical Experience .................................................................................................. 31
5.4 Clinical Experience ........................................................................................................ 31
6 Study Objectives .................................................................................................................. 31
6.1 Primary Objective ........................................................................................................... 31
6.2 Secondary Objectives ..................................................................................................... 32
6.3 Exploratory Objective .................................................................................................... 32
7 Study Endpoints ................................................................................................................... 32
7.1 Primary Endpoints .......................................................................................................... 32
7.1.1 Safety Endpoints ..................................................................................................... 32
7.2 Primary Efficacy Endpoint ............................................................................................. 32
7.3 Key Secondary Efficacy Endpoint ................................................................................. 32
7.4 Secondary Endpoints ...................................................................................................... 32
7.5 Exploratory Endpoints .................................................................................................... 33
8 Study Population.................................................................................................................. 33
8.1 Inclusion Criteria ............................................................................................................ 33
8.2 Exclusion Criteria ........................................................................................................... 34
9 Study Implementation ......................................................................................................... 36
9.1 Study Design .................................................................................................................. 36
9.1.1 Stage 1: Screening and Pre-mobilization Period .................................................... 36
9.1.1.1 Rescreening and Repetition of Screening Assessment(s) ................................ 37
9.2 Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection, CTX001
Manufacture and Disposition ......................................................................................... 37
9.3 Stage 3: Myeloablative Conditioning (Stage 3A) and Infusion of CTX001 (Day 1,
Stage 3B) ........................................................................................................................ 38
9.3.1 Stage 3A: Myeloablative Conditioning .................................................................. 38
9.3.2 Stage 3B: CTX001 Infusion (Day 1) ...................................................................... 39
9.4 Stage 4: Follow-up Through Engraftment and Up To 2 Years After CTX001 Infusion 39
9.4.1 Stage 4A: Post-infusion In-hospital Follow-up (Engraftment) .............................. 39

9.4.2 Stage 4B: Post-engraftment Follow-up .................................................................. 40

9.5 Data Monitoring Committee........................................................................................... 40
9.6 Steering Committee ........................................................................................................ 41
9.7 Endpoint Adjudication Committee ................................................................................. 41
9.8 Method of Assigning Subjects to Treatment Groups ..................................................... 41
9.9 Rationale for Study Elements ......................................................................................... 41
9.9.1 Study Design........................................................................................................... 41
9.9.1.1 Primary Efficacy Endpoint............................................................................... 42
9.9.2 Study Population..................................................................................................... 42
9.9.3 Dose ........................................................................................................................ 43
9.9.4 Study Duration ........................................................................................................ 43
9.10 Prior and Concomitant Treatments and Procedures ....................................................... 44
9.10.1 Prior Medications ................................................................................................... 44
9.10.2 Venous Access ........................................................................................................ 44
9.10.3 Prohibited medications ........................................................................................... 44
9.11 Administration ................................................................................................................ 44
9.11.1 RBC Transfusion .................................................................................................... 44
9.11.2 Mobilization and Apheresis .................................................................................... 44
9.11.2.1 Mobilization ..................................................................................................... 44
9.11.2.2 Apheresis Procedure......................................................................................... 45
9.11.3 Conditioning: Busulfan Administration ................................................................. 45
9.11.4 CTX001 Infusion .................................................................................................... 46
9.11.5 Post-CTX001 Infusion Infection Prophylaxis and Surveillance ............................ 46
9.12 Enrollment Suspension Criteria and Stopping Rules ..................................................... 46
9.12.1 Enrollment Suspension Criteria .............................................................................. 46
9.12.2 Stopping Rules for Individual Subjects .................................................................. 47
9.13 Removal of Subjects ....................................................................................................... 48
9.14 Replacement of Subjects ................................................................................................ 48
10 Study Drug Information and Management ....................................................................... 48
10.1 Preparation and Dispensing ............................................................................................ 48
10.1.1 Manufacture of CTX001 ........................................................................................ 48
10.1.2 Shipment of CTX001 Product to Treatment Site ................................................... 49
10.1.3 Storage of CTX001 Product ................................................................................... 49
10.1.4 CTX001 Infusion Procedures, Dose, and Administration ...................................... 49
10.2 Drug Accountability ....................................................................................................... 50
10.3 Disposal of Unused Drug ............................................................................................... 50
10.4 Blinding and Unblinding ................................................................................................ 50
11 Assessments .......................................................................................................................... 50
11.1 Subject and Disease Characteristics ............................................................................... 50
11.2 Transfusions ................................................................................................................... 50
11.3 Diffusing Capacity of the Lungs for Carbon Monoxide ................................................ 50
11.4 Imaging Assessments ..................................................................................................... 50
11.5 Blood for Biomarker Assessments ................................................................................. 51
11.5.1 Blood for Exploratory Research ............................................................................. 51
11.6 Bone Marrow Aspirate ................................................................................................... 51
11.7 Sperm and Oocyte Banking ............................................................................................ 52

11.8 Patient Reported Outcomes ............................................................................................ 52

11.8.1 Pain-Scale (11 point NRS) ..................................................................................... 52
11.8.2 EQ-5D-5L ............................................................................................................... 52
11.8.3 FACT-BMT ............................................................................................................ 52
11.8.4 PROMIS-Fatigue and -Cognitive Function ............................................................ 53
11.8.5 ASCQ-Me ............................................................................................................... 53
11.9 Mini-Mental State Examination ..................................................................................... 53
11.10 Safety .............................................................................................................................. 53
11.10.1 Adverse Events ....................................................................................................... 53
11.10.2 Clinical Laboratory Assessments ........................................................................... 53
11.10.3 Physical Examinations and Vital Signs .................................................................. 55
11.10.4 Weight/Height ........................................................................................................ 55
11.10.5 Electrocardiograms ................................................................................................. 55
11.10.6 Contraception and Pregnancy ................................................................................. 55
11.10.6.1 Contraception ................................................................................................... 55
11.10.6.2 Pregnancy ......................................................................................................... 57
12 Statistical and Analytical Plans .......................................................................................... 58
12.1 Sample Size and Power .................................................................................................. 58
12.2 Analysis Sets .................................................................................................................. 58
12.3 Statistical Analysis ......................................................................................................... 58
12.3.1 General Considerations........................................................................................... 58
12.3.2 Background Characteristics .................................................................................... 59
12.3.2.1 Subject Disposition .......................................................................................... 59
12.3.2.2 Demographics and Baseline Characteristics .................................................... 59
12.3.2.3 Prior and Concomitant Medications................................................................. 60
12.3.3 Efficacy Analysis .................................................................................................... 60
12.3.3.1 Analysis of Primary Variables ......................................................................... 60
12.3.3.2 Analysis of Secondary Efficacy Endpoints ...................................................... 60
12.3.3.3 Analysis of Exploratory Endpoints .................................................................. 61
12.3.4 Safety Analysis ....................................................................................................... 61
12.3.4.1 Adverse Events................................................................................................. 62
12.3.4.2 Clinical Laboratory Assessments ..................................................................... 62
12.3.4.3 Electrocardiogram ............................................................................................ 63
12.3.4.4 Vital Signs ........................................................................................................ 63
12.3.4.5 Physical Examination ....................................................................................... 63
12.3.5 Other Analysis ........................................................................................................ 63
12.3.6 Interim and Independent Data Monitoring Committee Analyses ........................... 63
12.3.6.1 Interim Analysis ............................................................................................... 63
12.3.6.2 Independent Data Monitoring Committee Analysis ........................................ 63
13 Procedural, Ethical, Regulatory, and Administrative Considerations ........................... 64
13.1 Adverse Event and Serious Adverse Event Documentation, Severity Grading, and
Reporting ........................................................................................................................ 64
13.1.1 Adverse Events ....................................................................................................... 64
13.1.1.1 Definition of an Adverse Event........................................................................ 64
13.1.1.2 Clinically Significant Assessments .................................................................. 64
13.1.1.3 Documentation of Adverse Events................................................................... 64

13.1.1.4 Adverse Event Severity .................................................................................... 65

13.1.1.5 Adverse Event Causality .................................................................................. 66
13.1.1.6 Study Drug Action Taken ................................................................................ 66
13.1.1.7 Adverse Event Outcome .................................................................................. 67
13.1.1.8 Treatment Given............................................................................................... 67
13.1.2 Serious Adverse Events .......................................................................................... 67
13.1.2.1 Definition of a Serious Adverse Event............................................................. 67
13.1.2.2 Reporting and Documentation of Serious Adverse Events .............................. 68
13.1.2.3 Expedited Reporting and Investigator Safety Letters ...................................... 69
13.2 Administrative Requirements ......................................................................................... 69
13.2.1 Ethical Considerations ............................................................................................ 69
13.2.2 Subject Information and Informed Consent ........................................................... 70
13.2.3 Investigator Compliance ......................................................................................... 70
13.2.4 Access to Records ................................................................................................... 70
13.2.5 Subject Privacy ....................................................................................................... 70
13.2.6 Record Retention .................................................................................................... 71
13.2.7 Study Termination .................................................................................................. 71
13.2.8 End of Study ........................................................................................................... 71
13.3 Data Quality Assurance .................................................................................................. 71
13.4 Monitoring ...................................................................................................................... 72
13.5 Electronic Data Capture ................................................................................................. 72
13.6 Publications and Clinical Study Report .......................................................................... 73
13.6.1 Publication of Study Results................................................................................... 73
13.6.2 Clinical Study Report ............................................................................................. 73
14 References ............................................................................................................................. 74
15 Protocol Signature Pages .................................................................................................... 78
15.1 Sponsor Signature Page .................................................................................................. 78
15.2 Investigator Signature Page ............................................................................................ 79
16 Appendices............................................................................................................................ 80
16.1 Busulfan PK Collection .................................................................................................. 80
List of Tables
Table 3-1 Study CTX001-121: Screening (Stage 1) ............................................................... 7
Table 3-2 Study CTX001-121: Pre-mobilization Period and Autologous CD34+ Stem
Cell Apheresis (Stage 1 and Stage 2) ...................................................................... 9
Table 3-3 Study CTX001-121: Myeloablative Conditioning and Infusion of CTX001
(Stages 3A/3B) ...................................................................................................... 11
Table 3-4 Study CTX001-121: Post-infusion Through Follow-up (Stage 4) ....................... 13
Table 3-5 Study CTX001-121: Transfusion Regimen .......................................................... 15
Table 9-1 Mobilization and Apheresis Timing ..................................................................... 45
Table 11-1 Laboratory Test Panels ............................................................................................. 54
Table 13-1 AE and SAE Recording ........................................................................................ 65
Table 13-2 Grading of AE Severity ........................................................................................ 66
Table 13-3 Classifications for AE Causality ........................................................................... 66
Table 13-4 Classifications for Study Drug Action Taken With Regard to an AE .................. 66
Table 13-5 Classifications for Outcome of an AE .................................................................. 67

List of Figures
Figure 1 Polypeptide Chains in Adult Hemoglobin and Fetal Hemoglobin ....................... 25
Figure 2 Fetal Hemoglobin Protection of RBC Sickling .................................................... 26
Figure 3 Schematic of the CRISPR-Cas9 Complex ............................................................ 28
Figure 4 CRISPR-Cas9-Mediated Genome Editing Strategies ........................................... 29
Figure 5 Therapeutic Hypothesis for BCL11A Gene Editing .............................................. 30
Figure 9-1 CTX001-121 Study Design................................................................................... 36
Figure 9-2 Mobilization and Apheresis Cycle ........................................................................ 38

List of Abbreviations
Abbreviation Definition
AE adverse event
Allo-HSCT allogeneic hematopoietic stem cell transplant
ALP alkaline phosphatase
ALT alanine transaminase
ANC absolute neutrophil count
ASCQ-Me Adult Sickle Cell Quality of Life Measurement System
AST aspartate transaminase
AUC area under the concentration versus time curve
bpm beats per minute
Cas9 CRISPR-associated 9 nuclease
CBC complete blood count
CI confidence interval
CRF case report form
CRISPR clustered regularly interspaced short palindromic repeats
crRNA crisprRNA
CSSCD Cooperative Study of Sickle Cell Disease
DLco lung diffusing capacity for carbon monoxide
DMSO dimethylsulfoxide
DMC data monitoring committee
DNA deoxyribonucleic acid
DSB double-strand break
EC ethics committee
ECG electrocardiogram
eCRF electronic case report form
EDC electronic data capture
EENT eyes, ears, nose, and throat
EQ-5D-5L EuroQol Quality of Life Scale
ETF early termination of Follow-up
FACT-BMT functional assessment of cancer therapy-bone marrow transplant
FDA Food and Drug Administration
FSH follicle-stimulating hormone
G-CSF granulocyte colony stimulating factor
GGT gamma-glutamyl transferase
GPS Global Patient Safety
GvHD graft-versus-host disease
GWAS genome-wide association studies

Hb hemoglobin
HBB human B globin
HBcAb hepatitis B core antibody
HbF fetal hemoglobin
HbS hemoglobin S
HBV hepatitis B virus
HCV hepatitis C virus
HDR homology-directed repair
hHSPCs human hematopoietic stem and progenitor cells
HIV-1 human immunodeficiency virus-1
HLA human leukocyte antigen
HSCT hematopoietic stem cell transplant
HU hydroxyurea
ICF informed consent form
ICH International Council for Harmonization
INR international normalized ratio
IRB institutional review board
IV intravenous, intravenously
LDH lactate dehydrogenase
LT-HSC long-term hematopoietic stem cells
LVEF left ventricular ejection fraction
M month
max maximum value
MCH mean corpuscular hemoglobin
MCHC mean corpuscular hemoglobin concentration
MCV mean corpuscular volume
min minimum value
MRA magnetic resonance angiography
MRI magnetic resonance imaging
mRNA messenger RNA
n number of subjects
NAT nuclei acid testing
NHEJ non-homologous end-joining

NSAIDs non-steroidal anti-inflammatory drugs
NSG NOD SCID gamma
nt nucleotide
PAM protospacer adjacent motif
PBMC peripheral blood mononuclear cells
PE physical examination
PI principal investigator
PK pharmacokinetic(s)
PROMIS Patient-Reported Outcome Measurement Information System
PROs patient-reported outcomes
PT prothrombin time
PTT partial thromboplastin time
q6h every 6 hours
QC quality control
QRS the portion of an ECG comprising the Q, R, and S waves, together representing
ventricular depolarization
QT QT interval
QTcF QT interval corrected by Fridericia’s formula
RBC red blood cell
RDW red blood cell distribution width
RNA ribonucleic acid
RNP ribonucleoprotein complex
RPR rapid plasma reagin
SAE serious adverse event
SAP statistical analysis plan
SCD sickle cell disease
SD standard deviation
sgRNA single-guide RNA
SOP standard operating procedure
SUSAR suspected, unexpected, serious adverse reaction
TDT transfusion dependent β thalassemia
TE treatment-emergent
tracrRNA trans-activating RNA
TRM transplant-related mortality
TRV tricuspid regurgitant jet velocity

ULN upper limit of normal
VAS Visual Analogue Scale
VOC vaso-occlusive crises
WBC white blood cell
WHODD World Health Organization Drug Dictionary
Glossary of Terms
Term Definition
Engraftment Engraftment is defined as absolute neutrophil count (ANC) ≥500/µL for 3 consecutive
days achieved within 42 days after CTX001 infusion and without use of unmodified
CD34+ (backup) cells.
Intended genetic Intended genetic modifications are defined as indels that modify the sequence of the
modifications erythrocyte-specific enhancer in intron 2 of BCL11A
Severe SCD Severe SCD is defined by the occurrence of at least 2 severe VOCs per year during the
2-year period before screening, while receiving appropriate supportive care (e.g. pain
management plan, hydroxyurea) as judged by the investigator.
Severe Severe VOC is defined as any 1 of the following events:
vaso-occlusive • Acute pain event that requires a visit to a medical facility and administration of pain
crises (VOC) medications (opioids or IV NSAIDs) or RBC transfusions
• Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate
associated with pneumonia-like symptoms, pain, or fever

5 INTRODUCTION
5.1 Background
5.1.1 Hemoglobinopathies
Hemoglobin (Hb), is a tetramer formed of 4-globin peptides, each tightly associated with a heme
group that contains an atom of iron. During gestation, the predominant form of hemoglobin is
fetal hemoglobin (HbF), which is composed of 2 α-globin chains and 2 γ-globin chains. Shortly
before birth, there is a switch from HbF to adult hemoglobin, which contains 2 α-globin and
2 β-globin polypeptide chains (Figure 1). The switch from HbF to adult hemoglobin is mediated
by a transcriptional switch from γ-globin to β-globin within the β-globin gene cluster located on
chromosome 11.
Figure 1 Polypeptide Chains in Adult Hemoglobin and Fetal Hemoglobin
Hemoglobinopathies are disorders caused by genetic defects that affect the production or
function of hemoglobin molecules. Two of the most common of the hemoglobinopathies are
sickle cell disease (SCD) and β-thalassemia.
5.1.2 Sickle Cell Disease
SCD is one of the most common monogenic disorders affecting millions of people.1 It is
estimated to affect over 100,000 individuals in the US and about 42,000 individuals in Europe.2
The most severe and prevalent form of SCD, referred to as sickle cell anemia, is an autosomal
recessive disease due to homozygous mutations in which a valine replaces a glutamic acid at
position 6 in the β-globin protein which leads to polymerization of deoxygenated hemoglobin
and red blood cell (RBC) sickling.
SCD is a chronic disease, characterized by recurrent acute VOC that lead to acute pain, chronic
hemolysis, anemia, progressive tissue injury, and organ dysfunction. The disease affects multiple
organs causing acute and chronic complications such as acute chest syndrome, stroke, priapism,
splenic sequestration, osteonecrosis, renal failure, pulmonary hypertension, liver disease, bone
damage, limited growth, increased susceptibility to infections, fatigue, and progressive cognitive
decline.3, 4
About 90% of children born with SCD in the US or EU will survive into adulthood, but their
lifespan is shortened by 2 to 3 decades compared to the general population with a median age of
death of approximately 40 to 50 years.5-9

Approved therapies to prevent complications of SCD include hydroxyurea (HU) in the US and
EU and L-glutamine oral powder in the US.10-12 These therapies reduce complications of SCD;
however, patients can still have breakthrough VOC. Furthermore, HU is not effective in all
patients, is not well-tolerated and has carcinogenic and teratogenic risks. Allogeneic
hematopoietic stem cell transplant (HSCT) is the only known cure for SCD but HSCT is only
available to about 20% of patients who have a matched donor13 and graft-versus-host disease
(GvHD) is a known risk. Therefore, there is significant unmet medical need for the treatment of
SCD.
Gene editing with CTX001 is intended to induce changes in the DNA sequence in the autologous
CD34+ hHSPC such that upon erythroid differentiation in the patient, the expression of γ-globin
is increased, leading to an increase in HbF expression levels in adult erythroid cells. The increase
in HbF is expected to ameliorate the clinical manifestations of SCD.
5.2 Therapeutic Rationale
The CRISPR-Cas9 editing therapeutic approach is to restore HbF production through editing of a
non-coding region in the BCL11A gene. BCL11A is a transcriptional silencer of γ-globin gene
expression and hence a negative modulator of HbF.
Several independent lines of evidence support the therapeutic hypothesis that an increase in HbF
will ameliorate the clinical manifestations of SCD:
• Analyses of the molecular interactions show that HbF can inhibit the abnormal
polymerization of hemoglobin S (HbS) that leads to RBC sickling (Figure 2).14, 15
Figure 2 Fetal Hemoglobin Protection of RBC Sickling

A) HbS Polymerization B) Prevention of HbS Polymerization by HbF
(A) HbS tetramers polymerize under deoxygenated conditions due to interactions between the mutated
valine at position 6 and a hydrophobic patch of an adjacent HbS tetramer at positions 85 to 88.
(B) The anti-sickling effect of HbF is due to the substitution of a less hydrophobic amino acid at position
87 (glutamine rather than a threonine), which disrupts the hydrophobic pocket and prevents the interaction
of HbS tetramers to form polymers.
• Natural history and observational data supports the protective role of HbF in the context of
naturally occurring variations:
o Infants with SCD are typically asymptomatic when their HbF levels are high, and only
become symptomatic when HbF synthesis is significantly reduced, typically at
approximately 6 to 9 months of age.14
o Populations that have polymorphisms associated with higher concentrations of HbF
(mostly in Saudi Arabia and Indian subcontinent) have milder forms of SCD.16

o Case reports and small studies show that people who are homozygous for the sickle
variant but also have high expression of HbF through adulthood (Hereditary Persistence
of Fetal Hemoglobin [HPFH]) have few or no SCD symptoms despite having HbS
concentration >60%.17 In addition, people who are homozygous for deletional HPFH type
1 or 2 and harbor large deletions including the β-globin genes are clinically unaffected
while they have 100% HbF.17,18
• HU, which is an approved pharmacologic therapy for the treatment of SCD, increases
production of HbF and ameliorates SCD symptoms.19
• Posthoc analyses of a randomized, placebo-controlled study of HU in SCD patients
(Multicenter Study of Hydroxyurea [MSH)] and an observational study of SCD patients
(Cooperative Study of Sickle Cell Disease [CSSCD]) show a strong association between
increased HbF and reduced pain rate.
5.2.1 CRISPR-Cas9 Gene Editing Approach
Gene editing using CRISPR-Cas9 can be used to create genetic modifications in CD34+ human
hematopoietic stem and progenitor cells (hHSPCs) with high specificity and frequency that will
result in a similar phenotype to the naturally occurring HPFH-associated variants, and are
expected to increase the production of HbF.
been repurposed as a RNA-guided DNA-targeting platform used for gene editing.20 It relies on
the DNA nuclease Cas9, and 2 noncoding RNAs-crisprRNA (crRNA) and trans-activating RNA
(tracrRNA)—to target the cleavage of DNA.
• crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through
Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA.
Changing the sequence of the 5’ 20nt in the crRNA allows targeting of the CRISPR-Cas9
complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain
a sequence match to the first 20nt of the single-guide RNA (sgRNA) if the target sequence is
followed by a specific short DNA motif (with the sequence NGG) called a protospacer
• TracrRNA hybridizes with the 3’ end of crRNA to form an RNA-duplex structure that is
bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex
which can then cleave the target DNA.
• Once the CRISPR-Cas9 complex is bound to DNA at a target site, 2 independent nuclease
domains within the Cas9 enzyme will each cleave one of the DNA strands 3 bases upstream
of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA
terminate in a base pair (called a blunt end).
For the molecular reagents used in CTX001 production, the 2 RNA molecules (crRNA and
tracrRNA) are joined by a 4nt linker loop to form a chimeric RNA called sgRNA named SPY101
(Figure 3). The CRISPR-Cas9 (sgRNA/Cas9) complex together forms a ribonucleoprotein
complex (RNP) in situ.

• Engraftment studies in an immunocompromised mouse model using zinc finger nucleases to

disrupt the erythroid lineage-specific enhancer of BCL11A in human CD34+ cells showed
successful multilineage differentiation into lymphoid and myeloid cell lineages.28
• Data from 3 patients with microdeletions in a region that contains the BCL11A gene
(2p15-p16.1) show that in humans, normal hematopoiesis can occur despite a significant
reduction in the levels of BCL11A expression. These 3 patients, who had BCL11A
haploinsufficiency and a large reduction in BCL11A expression across mononuclear and
erythroid cell populations, also showed a large increase in HbF levels (16%, 24%, and 30%).
However, a variety of hematologic parameters were normal, including blood counts,
lymphocyte subtypes (assessed in 2 patients), and concentration of immunoglobulins
(measured in 1 patient).
• Engraftment data from nonclinical studies show that disruption of the BCL11A erythroid
lineage-specific enhancer using SPY101-RNP-edited human CD34+ cells did not impact
multilineage differentiation into lymphoid and myeloid cell lineages.
Because the repair process uses NHEJ, the gene editing will generate indels within the
non-coding BCL11A erythroid lineage-specific enhancer on chromosome 2, thus down-
regulating BCL11A in erythroid precursors with no effect expected in other hematopoietic
lineages. This noncoding change is expected to reactivate γ-globin gene transcription, and
elevate HbF protein in RBCs (Figure 5).
Figure 5 Therapeutic Hypothesis for BCL11A Gene Editing
5.2.3 CTX001
CTX001 is a cellular product consisting of autologous CD34+ hHSPCs modified by
CRISPR-Cas9-mediated gene editing. The target of the CRISPR-Cas9 gene editing is the

erythroid lineage-specific enhancer region of the BCL11A gene located on intron 2 between
exons 2 and 3 on chromosome 2. The edits created with the highly specific guide, SPY101,
target a critical transcription factor binding site (GATA1) at the erythroid lineage-specific
enhancer region, (identifies as DNase I hypersensitive site +58, DHS+58) of the BCL11A
gene.28-30 The gRNA-Cas9 RNP (SPY101-RNP) introduces DSBs in DNA in a
sequence-dependent manner. Repair of the DSB by NHEJ results in DNA indels, intended to
disrupt GATA1 binding, thereby lowering BCL11A transcription, with concomitant increases in
γ-globin and HbF levels. Since, SPY101-RNP precisely targets the non-coding erythroid lineage-
specific enhancer region of the BCL11A gene and not the BCL11A coding sequence itself ,
SPY101-RNP is expected to modulate the levels of expression of the BCL11A gene and protein
in cells solely of the erythroid lineage and not affect non-erythroid hematopoietic lineages.
5.3 Nonclinical Experience
Details of the nonclinical efficacy and toxicology studies can be found in the Investigator’s
Brochure.
Briefly, in in vitro studies investigating CTX001, CRISPR-Cas9 gene editing at BCL11A gene
erythroid enhancer of healthy donor CD34+ cells led to an increase in γ/(γ+β)-globin mRNA
ratios of 0.41 (SD ± 0.15) and HbF mean percentage of HbF/(HbF+HbA) protein levels of 29%
(SD ± 11%). Mean allele editing frequency ranged was 80% (SD ± 6%) and was uniform across
subpopulation of CD34+ cells, including long-term hematopoietic stem cells (LT-HSC). Majority
of editing was bi-allelic.
In mice xenotransplantation studies, there was no difference in engraftment between NOD SCID
gamma (NSG) mice infused with CTX001 or control (mock or EGFP) edited CD34+ hHSPCs at
16 weeks post-transplantation. The average frequency of edited alleles present in the bone
marrow samples at 16 weeks was 91% (SD ± 15%). Furthermore, the engrafted cells were able to
maintain their multi-lineage potential.
In the nonclinical safety assessment of CTX001, on-target and potential off-target editing was
systematically evaluated using multiple orthogonal methods. CTX001 demonstrated high rate of
on-target indels (approximately 88%) and no off-target editing as compared to unedited healthy
donor cells. Karyotyping analysis did not identify any chromosomal translocations or other
detectable abnormalities.
Tumorigenicity study did not reveal any neoplastic or myeloproliferative lesions in the mice
receiving CTX001.
5.4 Clinical Experience
The current study will be the first-in-human study with CTX001 in subjects with SCD. CTX001
will also be evaluated in subjects with transfusion dependent β-thalassemia (TDT) in
Study CTX001-111. Currently, no subject has been dosed with CTX001.
6 STUDY OBJECTIVES
6.1 Primary Objective

• Evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9 modified
CD34+ hHSPCs (CTX001) in subjects with severe SCD

6.2 Secondary Objectives

• Assess the effects of infusion of CTX001 on disease-specific events and clinical status
• Quantify gene editing efficiency
6.3 Exploratory Objective
• Assess the ability of biomarkers to characterize CTX001 effect and predict treatment
outcomes
7 STUDY ENDPOINTS
7.1 Primary Endpoints

7.1.1 Safety Endpoints
• Successful neutrophil engraftment within 42 days after CTX001 infusion
• Safety and tolerability assessments based on adverse events (AEs), clinical laboratory values,
and vital signs
• Transplant-related mortality (TRM) within 100 days after CTX001 infusion
• TRM within 1 year after CTX001 infusion
7.2 Primary Efficacy Endpoint
• Proportion of subjects with sustained HbF ≥20% for at least 3 months starting 6 months after
CTX001 infusion, in the absence of treatment with HU
7.3 Key Secondary Efficacy Endpoint
• Relative change from baseline in annualized rate of severe VOC starting 6 months after
CTX001 infusion
7.4 Secondary Endpoints
• Reduction in annualized rate of severe VOC from baseline by at least 50%, starting 6 months
after CTX001 infusion
• Reduction in annualized rate of severe VOC from baseline by at least 65%, starting 6 months
• Absence of severe VOC events for at least 12 months at the time of the analysis
• Change from baseline in annualized duration of hospitalization for severe VOC, starting
6 months after CTX001 infusion
• Proportion of subjects with sustained HbF ≥20% for at least 3 months, starting 3 months after

• Proportion of subjects with sustained HbF ≥20% for at least 3 months, starting at the time of
• Change in number of units of RBC transfused for SCD-related indications over time
• HbF concentrations over time
• Hb concentrations over time
leukocytes over time
• Proportion of alleles with intended genetic modification present in bone marrow cells over
time
• Change in patient reported outcomes (PROs) over time using weekly Pain-Scale (11 point
numerical rating scale [NRS]), EuroQol Quality of Life Scale (EQ-5D-5L), functional
assessment of cancer therapy-bone marrow transplant (FACT-BMT), Patient-reported
Outcome Measurement Information System (PROMIS)-Fatigue, PROMIS-Cognitive
function, and Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me)
7.5 Exploratory Endpoints
• Change in hemolytic index as measured by principal component analysis of 4 markers of
hemolysis (reticulocyte count, and serum concentrations of aspartate transaminase, lactate
dehydrogenase [LDH], and total bilirubin) over time
• Change in tricuspid regurgitant jet velocity (TRV) over time
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) over
time
• Change in inflammatory and endothelial activation markers over time
8 STUDY POPULATION
Eligibility will be reviewed and documented by an appropriately qualified member of the

investigator’s team before subjects are enrolled.
Subjects who meet all of the inclusion criteria and none of the exclusion criteria will be eligible.
8.1 Inclusion Criteria
study:
1. Subject will sign and date an informed consent form (ICF).
2. Subjects 18 to 35 years of age, inclusive on the date of informed consent.
3. Documented βS/βS genotype. Subjects can be enrolled based on historical βS/βS genotype
result, but confirmation of genotype is required before busulfan conditioning.

4. Subjects with severe SCD. Severe SCD is defined by the occurrence of at least 2 of the
following events per year during the 2-year period before screening, while receiving
appropriate supportive care (e.g. pain management plan, HU) as judged by the investigator:
• Acute pain events that requires a visit to a medical facility and administration of
pain medications (opioids or intravenous [IV] non-steroidal anti-inflammatory
drugs [NSAIDs]) or RBC transfusions
• Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate
associated with by pneumonia-like symptoms, pain, or fever
8. Male subjects must agree to use effective contraception from start of mobilization through at
least 6 months after CTX001 infusion
10. Willing to participate in an additional long-term follow-up study after completion of this
study.
8.2 Exclusion Criteria
Subjects meeting any of the following criteria are not eligible for enrollment:
2. Prior HSCT.
4. White blood cell (WBC) count <3 × 109/L or platelet count <50 × 109/L, not related to
hypersplenism per investigator judgment.
5. Treatment with regular RBC transfusions that, in the opinion of the investigator, cannot be
interrupted after engraftment.
6. Subjects with history of alloimmunization to RBC antigens and for whom the investigator
anticipates that there will be insufficient RBC units available for the duration of the study.
7. More than 10 unplanned hospitalizations or emergency department visits related to SCD in
the 1 year before screening.
8. HbF level >15.0%, irrespective of concomitant treatment with HbF inducing treatments such
as HU.

9. History of untreated Moyamoya disease or presence of Moyamoya disease at Screening that

in the opinion of the investigator puts the subjects at the risk of bleeding.
confound the results of the study or pose an additional risk to the subject. This may include,
but is not limited to: history of relevant drug allergies; history of cardiovascular or central
nervous system disease; history or presence of clinically significant pathology; history of
mental disease, or history of familial cancer syndrome.
13. Advanced liver disease, defined as
a. Alanine transaminase (ALT) >3 × the upper limit of normal (ULN) or direct bilirubin
value >2 × ULN, or
b. Baseline prothrombin time (PT) (international normalized ratio [INR]) >1.5 × ULN, or
biopsy
15. Lung diffusing capacity for carbon monoxide (DLco) <50% of predicted value (corrected for
hemoglobin and/or alveolar volume).
16. Left ventricular ejection fraction (LVEF) <45% by echocardiogram.
18. Intolerance, contraindication, or known sensitivity to plerixafor or busulfan. Prior
anaphylactic reaction with excipients of CTX001 product (dimethylsulfoxide [DMSO],
dextran).
19. Positive serology for human immunodeficiency virus-1 (HIV-1) or human immunodeficiency
virus-2 (HIV-2), hepatitis B virus (HBV) (Hepatitis B core antibody [HBcAb] or nuclei acid
testing [NAT]), or hepatitis C virus (HCV) (NAT). Positive serology for syphilis or any other
infectious disease marker as required by local testing for cellular processing.
20. Participation in another clinical study with an investigational drug/product within 30 days of
screening.
21. Subjects who are not able to comply with the study procedures outlined in the protocol as
judged by the investigator.

9 STUDY IMPLEMENTATION
9.1 Study Design

This is a single-arm, open-label, multi-site, single-dose, Phase 1/2 study in subjects who have
severe SCD. The study will evaluate the safety and efficacy of a single dose of autologous
CRISPR-Cas9 modified hHSPCs (CTX001) and will initially include up to 12 subjects. The
subjects will be 18 to 35 years (inclusive) of age. The study may be expanded to include a total
of up to 45 subjects.
As a safety measure during the initial stages of the study, the first 2 subjects will be treated with
before treating the second subject in the study. The second subject will not undergo
myeloablation until the first subject achieves neutrophil engraftment (absolute neutrophil count
[ANC] ≥500/µL for 3 consecutive days), the available engraftment and safety data have been
reviewed by the data monitoring committee (DMC), and at least 30 days after infusion of
CTX001 to the first subject. Once the second subject has achieved neutrophil engraftment and
has not had Grade ≥3 AEs other than those typically associated with busulfan conditioning or
autologous transplant procedure, the remaining subjects can undergo conditioning and CTX001
infusion concurrently. In the event that the second subject infused with CTX001 experiences
Grade ≥3 AEs other than those typically associated with busulfan conditioning or autologous
transplant procedure, a DMC meeting will be convened and data will be reviewed before the
remaining subjects can undergo conditioning and CTX001 infusion. All steps that precede
busulfan myeloablation such as consent, screening, and stem cell collection may proceed
concurrently without staggering subjects.
The decision to expand the study to include a total of up to 45 subjects will be based on review
of available safety and efficacy data by the Sponsor in consultation with the DMC and the
Steering Committee (SC) after at least subjects have received CTX001 infusion.
For each subject, the study will be conducted in 4 stages (Figure 9-1), which are described
below. All subjects infused with CTX001 will be asked to enroll into an additional long-term
follow-up study.
Figure 9-1 CTX001-121 Study Design
9.1.1 Stage 1: Screening and Pre-mobilization Period

Screening Visit assessments are listed in Table 3-1 and assessment during the pre-mobilization
period are listed in Table 3-2.

The investigator (or an appropriate authorized designee at the study site) will obtain informed
consent from each subject. After informed consent, subject eligibility will be determined.
Fertility preservation
Subjects who meet the inclusion criteria will have the option to undergo fertility preservation via
cryopreservation of oocyte or sperm (this may occur at any time after eligibility is confirmed,
before initiation of busulfan conditioning).
RBC transfusion
After eligibility is confirmed, subjects will begin RBC transfusions (simple or exchange)
8 (± 2) weeks before the planned start of mobilization and will continue receiving these
transfusions until they begin busulfan conditioning. The goal of these RBC transfusions is to
target HbS level of <30% of total Hb while keeping total Hb concentration ≤11 g/dL. Treatment
with HU should be stopped at least 6 weeks before planned mobilization.
9.1.1.1 Rescreening and Repetition of Screening Assessment(s)
Individuals who do not meet the eligibility criteria for participation upon initial screening (screen
failure) can be rescreened once. Subject must be re-screened within 90 days of initial screen
failure. All screening tests should be repeated to determine eligibility except for: genotyping;
DLco and echocardiograph , if performed within 6 weeks unless judged by the investigator to be
necessary. Brain MRI/MRA should be repeated only if there are signs of neurological symptoms
as judged by the investigator.
screening process.
9.2 Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection,
CTX001 Manufacture and Disposition
Assessments during mobilization and apheresis are listed in Table 3-2.
Before starting administration of plerixafor, subjects will be assessed by the study investigator to
confirm whether they are eligible to proceed with apheresis (as per local guidelines). If a subject
is deemed to not be eligible for mobilization and apheresis, this procedure can be delayed for up
to 2 months. Thereafter, the subject will be removed from the study.
Each subject will undergo stem cell mobilization with plerixafor only (see Section 9.11.2.1 for
details). Peripheral blood mononuclear cells (PBMC) will be collected by apheresis.
On Day 1, subjects will receive plerixafor . Subjects will
undergo apheresis for 2 or 3 consecutive days to collect CD34 hHSPC (see Section 9.11.2.1 for
details). The targeted CD34+ cell collection is at least 15 × 106 CD34+ cells/kg for manufacturing
of CTX001 in order to achieve a minimum target dose of 3.0 × 106 CD34+ cells/kg. An
additional 2 × 106 CD34+ cells/kg will be collected as backup for rescue therapy in an event of
non-engraftment with CTX001. Cell collection intended for manufacturing will only occur on
Days 1 and 2 of apheresis. On these days collected cells intended for manufacturing will be
shipped daily at 2°C to 8°C to the manufacturing facility. Day 3 of apheresis is optional and
reserved for collection of backup cells ONLY. These cells will not undergo the
editing/manufacturing process and will be cryopreserved at the site. Backup cells may also be

begin RBC transfusions (simple or exchange) 8 (± 2) weeks before the planned start of busulfan
conditioning with the goal to maintain HbS level of <30% of total Hb while keeping total Hb
concentration ≤11 g/dL.
If HbS level is >30% of total Hb within 7 (±3) days before the planned start of busulfan
conditioning, subjects will receive 1 exchange transfusion with the goal to ensure HbS level is
<30% before start of busulfan conditioning (Table 3-5). After the CTX001 product is received at
the site and it has been confirmed that the backup cells remain available and in acceptable
condition to be administered if needed, the subject will be hospitalized.
Subjects will be assessed by the investigator to confirm their eligibility to proceed with
myeloablative conditioning before administration of busulfan. If a subject is deemed not to be
eligible for myeloablative conditioning, this procedure can be delayed for up to 2 months.
Thereafter, the subject will be removed from the study and the Sponsor will be notified.
The starting dose of busulfan will be 3.2 mg/kg administered IV once daily for 4 consecutive
days. However, busulfan may be administered as 0.8 mg/kg every 6 hours (q6h) for
4 consecutive days, per the site’s standard practice. Clinical sites may use a test dose of busulfan
30 (±2) days before beginning myeloablation to pre-determine busulfan dose.
The dose of busulfan may be adjusted as required based on the pharmacokinetics (PK) of the first
busulfan dose to maintain appropriate levels for myeloablation. Additional details regarding
busulfan administration are included in Section 9.11.3.
• CTX001 integrity has been compromised in any way and is no longer suitable for
• The subject has any clinical condition that, in the opinion of the investigator, would put
9.3.2 Stage 3B: CTX001 Infusion (Day 1)
CTX001 infusion will occur at least 48 hours after completion of busulfan infusion and no more
than 7 days after completion of busulfan infusion.
On Day 1, within 20 minutes after thawing, the entire dose of CTX001
(≥3 × 106 CD34+ cells/kg) will be infused through a central venous catheter. All vial(s)
containing CTX001 should be infused.
If the investigator suspects that CTX001 integrity has been compromised in any way and is no
investigator should not infuse CTX001 and immediately contact the medical monitor.
9.4 Stage 4: Follow-up Through Engraftment and Up To 2 Years After
CTX001 Infusion
9.4.1 Stage 4A: Post-infusion In-hospital Follow-up (Engraftment)
Subjects will be monitored in the transplant unit and receive supportive care according to
standard practices for subjects undergoing HSCT. Subjects will receive RBC transfusions to

maintain Hb ≥7 g/dL (Table 3-5) and platelet transfusion per institutional standards. Subjects
will be monitored for AEs and engraftment. Subjects will be discharged from the transplant unit
upon neutrophil engraftment (ANC ≥500/μL for 3 consecutive days) and stabilization of major
medical issues as per local hospital guidelines and investigator judgment. Details on post-
infusion infection prophylaxis and surveillance are included in Section 9.11.5.
9.4.2 Stage 4B: Post-engraftment Follow-up
Stage 4B starts after subjects have successfully engrafted, are clinically stable, and have been
discharged from the transplant unit. Subjects will be followed for approximately 2 years after
CTX001 infusion with physical examinations (PEs), laboratory and imaging assessments, and
AE evaluations.
Following engraftment, RBC transfusions should be avoided for hemoglobin ≥7 g/dL, unless
medically indicated (e.g., symptomatic anemia or as a requirement for surgery).
comprehensive PEs, and receive screening for malignancy based on appropriate country-specific
cancer guidelines and subject medical history. Subjects should also undergo appropriate
malignancy evaluation if they have unexplained symptoms, signs, or laboratory abnormalities
that could be related to an underlying malignancy. Examples include unexplained weight loss or
fever, lymphadenopathy, abnormal blood counts. The medical monitor should be notified if a
subject is diagnosed with a malignancy.
All subjects who receive CTX001 product will be asked to enroll in a long-term follow-up study
following completion or withdrawal/discontinuation.
9.5 Data Monitoring Committee
A DMC will be formed before the first subject is dosed. The DMC’s objectives and operational
details will be defined in a separate document (DMC charter). The DMC will be charged with
ensuring the safety of the subjects, safeguarding their interests, and ensuring the quality and
integrity of the trial. The DMC will conduct reviews of study data as outlined in the DMC
charter. The DMC may recommend that the Sponsor suspend enrollment, amend the study, or
The DMC will review available engraftment and safety data for the first subject before the
second subject will undergo myeloablation. The DMC will also review data from the second
subject if the second subject infused with CTX001 experiences Grade ≥3 AEs other than those
typically associated with busulfan conditioning or autologous transplant procedure, before the
remaining subjects can undergo myeloablation and CTX001 infusion.
CTX001 in order to recommend whether or not to expand the study to include up to 45 subjects
(expansion cohort).

9.6 Steering Committee

9.7 Endpoint Adjudication Committee
group of experts with appropriate clinical and scientific background to evaluate VOC events. The
EAC will be adjudicating VOC events to ensure that the VOCs used for determining the baseline
and on-study rate of VOCs meet the definition of the study defined severe VOC. The EAC will
not be adjudicating VOCs for determining study eligibility.
9.8 Method of Assigning Subjects to Treatment Groups
This is an open-label study.
9.9 Rationale for Study Elements
9.9.1 Study Design
The study is single-arm and open-label because of the need for stem cell transplant procedure to
deliver CTX001.
The overall process is consistent with procedures used for autologous HSCT in patients with
malignant diseases, with a few exceptions that are described below. Therefore, the risk
associated with the procedures in this study is not expected to be significantly different from the
standard risks of these procedures.
Prophylactic RBC transfusions (simple or exchange) will be administered starting 8 (±2) weeks
before mobilization through CTX001 infusion, to decrease the risk of VOC during mobilization
and busulfan conditioning. The rationale for this transfusion guidance stems from evidence
demonstrating that transfusion of non-sickle RBCs by simple or exchange techniques into
patients with SCD can mitigate physiologic complications such as stroke31, disease-related
complications after surgical procedures32, ACS33, and VOCs.34 Stem cell transplant protocols for
patients with SCD often involve exchange transfusion to decrease HbS <30% prior to
mobilization and conditioning.
Following mobilization, CD34+ stem cells will be collected by apheresis with plerixafor alone.
Collection of stem cells by apheresis rather than bone marrow harvest allows for easier isolation
of CD34+ cells as the process is less invasive for patients and does not require general anesthesia.
The decision to use plerixafor alone instead of plerixafor in combination with granulocyte
colony-stimulating factor (G-CSF), which is the standard regimen used for collection, comes
from the fact that administration of G-CSF can induce severe VOCs in subjects with SCD, which
can be fatal.35 Data from studies in healthy subjects and subjects with β-thalassemia show that
plerixafor alone can effectively mobilize CD34+ cells.36,37, 38 These studies also show that CD34+
cells collected using plerixafor alone in healthy subjects successfully engraft, when administered
in the context of an allogeneic HSCT. In addition, a small study describing stem cell
mobilization with plerixafor alone in subjects with severe SCD reports the collection of a median
of 10.4 × 106 CD34+ cells/kg (5.1 to 20.0) after a single apheresis. However, a non-severe pain
crisis occurred in 3 of 7 subjects, despite administration of RBC transfusions in the preceding

is the only known cure for SCD but <20% of patients have a matched related donor and GvHD
remains a significant risk. Therefore, there is significant unmet medical need for the treatment of
patients with severe SCD.
This study will enroll subjects with severe SCD. Severe SCD is defined by the occurrence of at
least 2 of the following events each year during the 2-year period before screening, while
receiving appropriate supportive care (e.g., pain management plan, HU if indicated) as judged by
the investigator:
• Acute pain event that requires a visit to a medical facility and administration of pain
medications (opioids or IV NSAIDs) or RBC transfusions
• Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate associated
with pneumonia-like symptoms, pain, or fever
Recurrent VOC events will serve as a clinical endpoint to allow assessment of the clinical
benefits by assessing the change in severe pain crises and other VOC manifestations.
Subjects with baseline HbF >15% will not be eligible for enrollment. Since very few patients
with SCD who have high HbF levels have recurrent severe VOC, an upper limit for HbF was
included to support the fact that the events collected from the medical records are acute severe
VOC related to SCD. It will also ensure that subjects meeting the primary efficacy endpoint will
have a meaningful increase in HbF levels.
Subjects who have a history of requiring regular RBC transfusions to prevent SCD complications
will not be eligible for enrollment because in order to accurately assess the primary efficacy of
“HbF ≥20% for at least 3 months,” RBC transfusion will have to be stopped after CTX001
infusion as specified per study protocol.
9.9.3 Dose
Autologous transplantation for various indications typically uses at least 2 to 2.5 × 106
CD34+ cells/kg to support engraftment.47-49 To ensure engraftment in all subjects in the SCD
study, the Sponsor selected a conservative minimum dose of 3 × 106 CD34+ cells/kg, which is
20% to 50% higher than the typical minimum dose for autologous transplantation. In principle,
infusion of a higher number of CD34+ stem cells after myeloablation is associated with more
rapid engraftment, durability, and efficacy of the treatment.50
Based on current manufacturing capabilities and projected cell yields, the Sponsor proposes to
set a maximum cell dose limit of 2 × 107 CD34+ cells/kg. The Sponsor, along with the DMC,
will monitor for any potential dose related toxicities.
9.9.4 Study Duration
Subjects will be followed on study for 2 years after CTX001 infusion. A 2-year follow up
duration was considered adequate for characterization of clinical outcomes. Because the study
will enroll subjects with at least 2 severe VOC events per year during the 2-year period before
screening, a 2-year follow-up will allow meaningful assessment of the decrease in the annualized
incidence of VOC.

follow-up study for up to 15 years following completion or withdrawal/discontinuation. This is
to ensure that any potential long-term AEs related to CTX001 are captured and to evaluate
long-term treatment outcomes as well as provide a longer follow up for efficacy.
9.10 Prior and Concomitant Treatments and Procedures
9.10.1 Prior Medications
All medication taken within 30 days of screening will be recorded.
Retrospective information on RBC transfusions will be recorded from 24 months prior to date of
consent study screening.
9.10.2 Venous Access
infusion of CTX001. A central venous catheter may also be used for apheresis, exchange
transfusions, and for clinical care of the subject following CTX001 infusion as per investigator
judgment.
9.10.3 Prohibited medications
HU: Treatment with HU should be discontinued at least 6 weeks before planned start of
mobilization. Subjects may be restarted on HU after mobilization and apheresis if deemed
necessary by the investigator and if >4 months are planned between completion of mobilization
and start of busulfan conditioning. If HU is restarted, treatment with HU should be discontinued
once RBC transfusions are restarted before busulfan conditioning.
After CTX001 infusion, once engraftment is achieved, HU may be restarted if the subject
experiences VOCs or other complications that are judged by the investigator to be related to
SCD and warrant re-initiation of HU. If HU is restarted, it is recommended that HU be
progressively tapered, if considered medically acceptable and if HbF is ≥20%.
L-Glutamine: Treatment with L-Glutamine should be discontinued after CTX001 infusion.
HbF inducing agent (other than HU): Treatment with any HbF inducing treatment should be
discontinued after CTX001 infusion.
9.11 Administration
9.11.1 RBC Transfusion
Refer to Table 3-5 for details on RBC transfusion.
9.11.2 Mobilization and Apheresis
9.11.2.1 Mobilization
Decision on whether a central line is needed for mobilization will be made by the
apheresis-experienced nurse or physician. Mobilization will be with plerixafor only. G-CSF
should NOT be administered.
Subjects will receive plerixafor at a dose of 0.24 mg/kg via subcutaneous injection
. Weight taken within 5 days before the first day of mobilization will be
used for calculating the recommended dose. Refer to Table 9-1 for full dosing schedule.

CD34+ cell count in peripheral blood will be performed as listed in Table 3-2.
Refer to the plerixafor package insert for product details. Refer to the study reference manual for
further details on mobilization, apheresis, and infusion procedures.
Table 9-1 Mobilization and Apheresis Timing
Drug Day 1 Day 2 Day 3

Plerixafor administration X X Xa
CD34+ stem cell apheresis for CTX001 product X X Xb

and backup cells
a
Only subjects undergoing apheresis on Day 3, should receive plerixafor on Day 3
b
The third day of apheresis is reserved ONLY for collection of backup cells. No collection of cells for
manufacturing should occur on that day.
9.11.2.2 Apheresis Procedure

PBMC will be collected per clinical site standard operating procedures (SOPs) for up to
3 consecutive days. The targeted CD34+ cell collection is at least 15 × 106 CD34+ cells/kg for
manufacturing of CTX001. An additional 2 × 106 CD34+ cells/kg will be collected as backup for
rescue therapy in an event of non-engraftment with CTX001. Collection of backup cells must
occur prior to proceeding to Stage 3 (busulfan conditioning).
CTX001 product and safety backup, or if a subject cannot complete apheresis due to VOC(s),
2 additional mobilization and apheresis cycles will be allowed to collect additional cells.
The additional mobilization cycle should be initiated at least 14 days after the first day of the
prior mobilization cycle and no more than 60 days after the end of the prior cycle. Based on the
number of CD34+ cells received at the manufacturing site and the manufactured CTX001 dose,
the medical monitor will inform the clinical site if additional mobilization cycle will be
necessary. The investigator should contact the medical monitor if the minimal cell dose for
manufacturing or backup is not reached or if the subject requires an additional mobilization
cycle.
Any AEs associated with apheresis procedure should be managed as per the site’s standard
guidelines.
Cells should be shipped at the end of the first and the second collection day. Cells collected for a
backup dose will be cryopreserved and stored at the study site. Additional details and
instructions on shipment and receipt are included in the study reference manual.
9.11.3 Conditioning: Busulfan Administration
Busulfan will be administered IV daily at a starting dose of 3.2 mg/kg/day for 4 consecutive days
(based on weight collected within 3 to 7 days prior to the first day of busulfan administration).
Once daily dosing is the preferred schedule, but the busulfan dose regimen may be adjusted to be

given q6h per site’s standard practice. A test dose of busulfan may be performed 30 (±) 2 days
before beginning myeloablation to pre-determine busulfan dose.
The dose of busulfan may be adjusted based on the PK of the first busulfan dose to maintain
appropriate levels for myeloablation. The average target AUC for subjects at a starting dose of
3.2 mg/kg/day for 4 days is 5000 µM⋅min (range: 4500 to 5500); equivalent to target cumulative
busulfan exposure of 90 mg⋅hr/L (range 80-100 mg ⋅ hr/L). The AUC for subjects administered
busulfan q6h for 4 days is 1125 µM⋅min (range: 900 to 1350). Refer to Appendix 16.1 for
busulfan PK collection schedule.
9.11.4 CTX001 Infusion
CTX001 will be supplied in infusion vial(s). All vial(s) containing CTX001 should be infused.
Further description is provided in the reference manual.
9.11.5 Post-CTX001 Infusion Infection Prophylaxis and Surveillance
In the event that the subject develops sepsis or systemic bacteremia following CTX001 infusion,
appropriate cultures and medical management will be initiated.
9.12 Enrollment Suspension Criteria and Stopping Rules
9.12.1 Enrollment Suspension Criteria
• Death (non-accidental) on study after CTX001 infusion.
• Detection of leukemia, myelodysplastic disease, or myeloproliferative disorder in a
subject who has received CTX001.
• Failure to achieve neutrophil engraftment after infusion of CTX001 in 2 subjects, as
cells.
• Any serious adverse event (SAE) at least possibly related to CTX001 in 3 subjects, with
the exception of
o Grade 3 elevation in ALT, aspartate transaminase (AST), alkaline phosphatase
(ALP), gamma-glutamyl transferase (GGT), or bilirubin that resolve to baseline or
within normal range in 10 days

• Occurrence of VOC associated with a Grade 4 treatment-related AE in 2 subjects, during

or immediately after stem cell mobilization and collection.
• Failure to collect target number of CD34+ cells in 3 of the first 6 subjects.
Enrollment will be temporarily suspended for lack of efficacy if:

• HbF is <5% at 9 months in 3 of the first 12 subjects
• The proportion of alleles with intended genetic modification present in peripheral blood
leukocytes is <10% in 3 of the first 12 subjects at 2 consecutive time points after
will be reviewed. After evaluation, and following a review of the data with the DMC, the
Sponsor and the SC may decide to restart enrollment in the study, amend the study, or
permanently suspend enrollment and remove all subjects who have not received CTX001 from
the study. A substantial amendment will be submitted before resuming enrollment.
Sponsor and the SC may decide to restart enrollment, amend the study, or permanently suspend
followed up or transitioned to a long-term safety follow-up study.
unedited CD34+ cells will be up to the investigator’s discretion after discussion of the potential
risks with the subject. The investigator should inform the medical monitor of the decision before
infusion of cellular product.
parties including principal investigators (PIs), Ethics Committees (EC), IRB, and competent
authorities.
9.12.2 Stopping Rules for Individual Subjects
risk for continuing study-related procedures or follow-up
• Inability to successfully collect the target number of CD34+ cells
before the start of busulfan conditioning
• Failure to successfully manufacture CTX001

9.13 Removal of Subjects

A subject (or legally authorized representative) may withdraw consent to participate in the study
at any time. If a subject withdraws consent, the date and reason for withdrawal should be
documented. Subject data collected up to the date of consent withdrawal will be included in the
analyses. If the subject withdraws consent for the study, no further evaluations will be
performed, and no additional samples will be collected. The Sponsor may retain and continue to
use any data and samples collected before such withdrawal of consent. Wherever possible, the
tests and evaluations listed for the end of study visit will be carried out. The Sponsor will be
notified of all study withdrawals.
If a subject withdraws consent to participate in the study after infusion of CTX001, the subject
will be asked to enroll in the long-term follow-up study. If the subject refuses to participate in the
long-term follow-up study, the subject will be asked if they consent to have their information
collected through review of medical records or quarterly telephone interviews (partial
withdrawal of consent). All subjects who withdraw after CTX001 infusion should be followed
for safety whenever possible.
Other reasons for removal of a subject before start of busulfan conditioning include:
• Any medical condition that, in the opinion of the investigator, would put the subject at risk
for continuing study-related procedures or follow-up
• Subject becomes pregnant or begins breastfeeding
• Failure to successfully manufacture the minimum dose of CTX001 or deliver CTX001 to the
site
• Administrative decision by Sponsor
If a subject chooses to withdraw from the study after the start of busulfan conditioning and
before administration of CTX001, the subject’s stored, unedited backup stem cells will be
If a 10/10 HLA matched related donor becomes available after enrollment but before starting
busulfan conditioning, then the subject will be withdrawn from the study.
9.14 Replacement of Subjects
10 STUDY DRUG INFORMATION AND MANAGEMENT
10.1 Preparation and Dispensing

10.1.1 Manufacture of CTX001

cryopreservation in the vapor phase of liquid nitrogen (≤-135°C). QC testing and batch release
should normally be completed in . Product will then be shipped at ≤-135°C to the clinical
site in 1 or more 20 ml vials. All manufacturing procedures are carried out in GMP cleanrooms.
10.1.2 Shipment of CTX001 Product to Treatment Site
CTX001 (packaged in vial[s]) will be shipped cryopreserved to the clinical sites in sealed dry
nitrogen shippers (≤-135°C) validated for at least 10 days. On receipt, package integrity will be
10.1.3 Storage of CTX001 Product
CTX001 will be stored at the site in the frozen state at a temperature of ≤-135°C until just prior
manual.
10.1.4 CTX001 Infusion Procedures, Dose, and Administration
Dextran-40. Histamine release associated with DMSO can result in symptoms such as; adverse
effects including nausea, vomiting, diarrhea, flushing, fevers, chills, headache, dyspnea, rashes,
bronchospasm, anaphylaxis, vasodilation and hypotension, and mental status changes. Given the
risk of these AEs, subjects will be pre-medicated with an antihistamine (diphenhydramine
hydrochloride) and an antipyretic (acetaminophen or paracetamol) before dosing with CTX001.
These medications may be repeated every 3-4 hours as needed as per institutional guidelines and
site SOPs. The entire dose of CTX001 should be infused within 20 minutes of thaw. Detailed
instructions for the thaw and infusion of cells are in the study reference manual. Hospital
guidelines will be used to maintain chain of custody for CTX001 from the stem cell laboratory to
the subject. Before infusion, local procedures should be followed regarding the verification of
subject identity and product details to ensure a match as well as integrity of the product.
In the unlikely event that CTX001 infusion does not occur within 7 days after the last dose of
busulfan, subjects should receive the backup CD34+ stem cells.

10.2 Drug Accountability

10.3 Disposal of Unused Drug
10.4 Blinding and Unblinding
11 ASSESSMENTS
The schedule of assessments is shown in Table 3-1, Table 3-2, Table 3-3, Table 3-4, and
Table 3-5.
11.1 Subject and Disease Characteristics
Demographics (e.g., sex, age, race, ethnicity), 2 years of transfusion history (simple or exchange
transfusions), and 2 years of SCD-related complications and associated treatment (including in-
patient hospitalization and medical facility visits) will be recorded. SCD history such as
genotype and age of diagnosis will also be recorded. Past and present medical history, including
targeted medical history and additional medical history considered by the investigator to be
significant will also be recorded.
11.2 Transfusions
Any transfusion received and reason for transfusion (mL and number of transfused units) will be
documented from the time of consent through the end of the study. Additionally, Hb
concentration before transfusion will be documented.
11.3 Diffusing Capacity of the Lungs for Carbon Monoxide
DLco (corrected) will be assessed. Pulmonary function tests will be performed with the subject
in a seated position and should be performed per site’s SOPs for pre-transplant workup and using
a calibrated spirometer.
11.4 Imaging Assessments
A brain MRI/MRA will be performed (for assessment of Moyamoya disease) at screening. If a
brain MRI/MRA has been performed within 3 months before date of consent, these results may
be used, unless repetition of the assessment is clinically indicated.

A transthoracic cardiac echocardiograph (for assessment of LVEF and TRV) will be performed
and read by trained medical personnel. Subjects who develop signs/symptoms of congestive
heart failure at any point during the study will undergo LVEF measurement by echocardiograph.
Detailed procedures for image acquisition and analysis will be provided in a separate document.
11.5 Blood for Biomarker Assessments
Blood samples will be collected for evaluation of biomarkers and their ability to characterize the
effect of CTX001 and predict treatment outcomes.
quantitation in peripheral blood to assess bulk HbF levels, (2) total hemoglobin
concentration, (3) the proportion of circulating erythrocytes expressing fetal hemoglobin
(F-cells) (4) inflammatory (CRP, IL6, IL8) and endothelial activation markers (E-
Selectin, P-Selectin) 1
leukocyte DNA.
11.5.1 Blood for Exploratory Research
Blood samples will be collected for potential exploratory research to better understand CTX001
and its impact on hemoglobinopathies.
Samples may be used for exploratory research to identify molecular (genomic, metabolic and/or
proteomic) biomarkers that may be indicative of clinical response, resistance, safety,
pharmacodynamic activity (e.g. anti-Cas9 antibodies in cases of significant [>50%] HbF
decrease) and/or the mechanism of action of treatment. Samples of apheresed stem cells and
CTX001 will be collected during manufacturing for potential future genome analysis in case of
development of malignancy thought to be related to CTX001 and for exploratory research to
better understand CTX001 and its impact on SCD. Samples will not impact a subject’s minimum
final CTX001 dose.
11.6 Bone Marrow Aspirate
Bone marrow aspirate samples will be collected for allelic editing testing to assess the proportion
of alleles with intended genetic modification present in bone marrow DNA.
Samples may also be used for potential future genome analysis in case of development of
malignancy thought to be related to CTX001. Additionally, samples may be used for exploratory
research to identify molecular (genomic, metabolic and/or proteomic) biomarkers that may be
indicative of clinical response, resistance, safety, pharmacodynamic activity and/or the
mechanism of action of treatment.

Detailed procedures for processing and handling bone marrow aspirate samples will be provided
in a separate document.
11.7 Sperm and Oocyte Banking
choose to undergo the procedures will do so after eligibility is confirmed and prior to busulfan
conditioning (Stage 3A).
11.8 Patient Reported Outcomes
PRO assessments should be performed as the first assessment after obtaining informed consent,
as well as be completed by subjects at the beginning of a study visit prior to any assessments.
11.8.1 Pain-Scale (11 point NRS)
Numerical rating scale is a 1-dimensional measure of reporting intensity of pain in adults. The 11
point NRS is a segmented visual analogue scale (VAS) including numbers from 0 to 10, ‘0’
representing no pain to ‘10’ representing worst possible pain. Each respondent will select a
whole number on the scale that reflects their pain intensity. The score of NRS ranges from 0 to
10 points, with higher values indicating a higher level of pain.
11.8.2 EQ-5D-5L
The EQ-5D-5L questionnaire assesses a subject’s health status in a standardized way and
consists of 2 parts: the EQ-5D descriptive system and the EQ VAS.
The EQ-5D-5L descriptive system comprises the same 5 dimensions; mobility, self-care, usual
activities, pain/discomfort, and anxiety/depression. Each dimension has 5 levels: no problems,
The EQ VAS records the subject’s self-rated health on a 100-point VAS with endpoints labeled
11.8.3 FACT-BMT
The FACT-BMT questionnaire is a validated self-report questionnaire that includes physical,
social, family, emotional, and functional well-being. The FACT-BMT consists of the FACT-
General (constitutes the core of all subscales) and treatment-specific concerns of bone marrow
transplantation.
Each statement in the FACT-BMT has a 5 point Likert-type response scale ranging from 0 to 4
(0 = “not at all”; 1 = “a little bit”; 2 = “somewhat”; 3 = “quite a bit”; and 4 = “very much”). The
subject is asked to circle or mark 1 number per line to indicate his/her response to the statement
as it applies to the past 7 days. Questionnaires are then scored, and the higher the score, the
better the QOL.

11.8.4 PROMIS-Fatigue and -Cognitive Function
PROMIS item bank contains over 300 items of illness-related PROs. PROMIS-Fatigue 7a
contains 7 items and evaluates self-reported symptoms such as feeling tired and extreme
exhaustion, and the impact of these symptoms on daily activities and normal functioning.
PROMIS-Cognitive function 8a contains 8 items pertaining to the ability to think, concentrate,
etc. Both measures have a 7-day recall period (“in the past 7 days”). Each question has 5 levels:
never, almost never, sometimes, often, and almost always. PROMIS measures are scored using
T-score metric with a mean of 50 for reference population and SD of 10. Scores can be
interpreted by considering the direction of scoring and the difference between the reported score
and the mean of 50 in reference population (SD 10).
11.8.5 ASCQ-Me
ASCQ-Me comprises of measures to assess physical, mental and social health along with
information on severity of disease. It includes the following domains: emotional impact, pain
impact, pain episodes, sleep impact, social functioning impact, stiffness impact and SCD medical
history checklist. Most dimensions have 5 levels: never, rarely, sometimes, often, and always or
not at all, a little bit, somewhat, quite a bit, and very much. Questions on SCD medical history
checklist are indicated by yes or no options and pain episode frequency and severity are
indicated by frequency of events. ASCQ-Me domains are scored using T-score metric with mean
of 50 for reference population and SD of 10. Scores can be interpreted by considering the
direction of scoring and the difference between the reported score and the mean of 50 in
reference population (SD 10).
11.9 Mini-Mental State Examination
Subjects will be evaluated for cognitive function using the Mini-Mental State Examination
(Version 2) after eligibility is confirmed and before start of transfusions.
11.10 Safety
Safety evaluations will include engraftment, TRM, AEs, clinical laboratory assessments, clinical
evaluation of vital signs, ECGs, and PEs.
11.10.1 Adverse Events
Section 13.1 outlines the definitions, collection periods, criteria, and procedures for
11.10.2 Clinical Laboratory Assessments
Blood and urine samples will be collected according to Table 3-1 through Table 3-4. Additional
clinical laboratory tests may be obtained at any time as clinically necessary for safety purposes at
the investigator’s discretion.

Females of childbearing potential (as defined in Section 11.10.6) must have a negative
pregnancy test result at screening, within 3 days prior to starting mobilization, and within 5 days
before the initiation of busulfan conditioning.
The laboratory test panels are shown in Table 11-1.
Table 11-1 Laboratory Test Panels

Hematology Serum Chemistry Urinalysis Other Tests
CBC with differential ALT Protein At screening only: Genotyping of HBB

including: AST Blood and alpha loci (central laboratory)
Hemoglobin Bilirubin (total and Nitrite
WBC direct) Leukocyte Hemoglobin fractionation (central
ANC Albumin esterase laboratory)
Platelet count Alkaline phosphatase
Reticulocyte count Bicarbonate Allelic editing (blood; central laboratory)
Nucleated RBCs BUN urea
MCV (Mean Calcium Allelic editing (bone marrow aspirate;
corpuscular volume) ChlorideCreatinine central lab)
MCHC (Mean Glucose (fasting)
corpuscular Magnesium HbF distribution, F-cells (central
hemoglobin Phosphorous laboratory)
concentration) Potassium
MCH (Mean Sodium CD34+ cell count
corpuscular Total protein
hemoglobin) Inflammatory and endothelial activation
RDW (Red blood cell markers (central laboratory)
distribution width)
If applicable:
Serum pregnancy test
Infectious Disease Immunological Testing Coagulation Hemolysis Marker
Marker Testing
HBV (core Ab and CD4 PT (Prothrombin Haptoglobin
NAT) CD8 time) Lactate dehydrogenase
HCV (NAT) CD19 aPTT (Activated
HIV-1, HIV-2 CD16 partial
(Antigen/Antibody CD56 thromboplastin
immunoassay, RNA) IgG time)
Syphilis (RPR, if IgD INR
reactive FTA-ABS IgE
[Rapid plasma reagin/ IgM
fluorescent IgA
treponemal antibody
absorption test])

11.10.3 Physical Examinations and Vital Signs

A PE includes a review of the following systems: head, neck, and thyroid; eyes, ears, nose, and
throat (EENT); respiratory; cardiovascular; lymph nodes; abdomen (including spleen); skin;
musculoskeletal; neurological systems, and Karnofsky performance status. Breast, anorectal, and
genital examinations will be performed when medically indicated. After screening, any clinically
significant abnormal findings in PEs will be reported as AEs.
The abbreviated PE will include an assessment of the following body systems: head, neck, and
thyroid; EENT; cardiovascular system; respiratory system; skin; and abdomen (including
spleen).
Vital signs include blood pressure (systolic and diastolic), temperature, pulse rate, pulse
oximetry, and respiration rate. The subject will be instructed to rest for at least 5 minutes before
vital signs are assessed.
11.10.4 Weight/Height
Subject weight (kg) and height (cm) will be measured with shoes off.
11.10.5 Electrocardiograms
Standard 12-lead ECGs will be performed using a machine with printout. Additional standard
12-lead ECGs will be performed at any other time if clinically indicated. The performance of all
ECGs will adhere to the following guidelines:
• The ECG will be done before any other procedures that may affect heart rate, such as blood
draws.
• The subject will be instructed to rest for at least 5 minutes before having an ECG.
• The test should be performed in the supine position.
A printout of the ECG traces will be made for safety review by the investigator and maintained
with source documentation. Clinically significant ECG abnormalities occurring during the study
through the Safety Follow-up Visit will be recorded as AEs.
11.10.6 Contraception and Pregnancy
11.10.6.1 Contraception
infusion.
Non-sterile male subjects who are or may become sexually active with female partners of
childbearing potential must agree to use acceptable, highly effective methods of contraception to
avoid fathering a child from start of mobilization through at least 6 months after CTX001
infusion.

Acceptable methods of contraception for subjects and their partners are listed below. If
applicable, additional contraception requirements may need to be followed according to local
regulations and/or requirements.
• True abstinence for the subject, when this is in line with the preferred and usual lifestyle of
the subject. Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation
methods) and withdrawal are not acceptable methods of contraception.
• If the male is infertile (e.g., bilateral orchiectomy). Infertility may be documented through
examination of a semen specimen or by demonstration of the absence of the vas deferens by
ultrasound before mobilization.
Female Subjects
• Female bilateral tubal ligation performed at least 6 months previously.
• Female continuous use of an intrauterine device (non-hormone releasing or hormone
• Female combined (estrogen and progestogen-containing) or progestogen-only oral hormonal
contraception associated with inhibition of ovulation if successfully used for at least 60 days
before mobilization or with a second form of approved contraception for at least 60 days
after beginning hormonal contraception.
Male Subjects
contraception.
• Hormonal contraceptives, if successfully used for at least 60 days before start of
mobilization

Additional notes:
• Female condom cannot be used with male condom (as a double method of contraception)
• The use of birth control methods does not apply if the female partner has had a bilateral
• Male subjects who are not sexually active at the time of screening must agree to follow
• If applicable, additional contraception requirements may need to be followed according
• Male subjects must not donate sperm after start of mobilization, throughout the study,
• Unique situations that may not fall within the above specifications may be discussed with
Female Subjects of Non-childbearing Potential:
criteria:
• Postmenopausal: Female subjects who have been amenorrheic for at least 2 years and have a
serum follicle-stimulating hormone (FSH) level within the laboratory's reference range for
postmenopausal females
• Documented hysterectomy and/or bilateral oophorectomy
Note: All other female subjects (including subjects with tubal ligations and subjects who do not
have a documented hysterectomy) will be considered to be of childbearing potential.
11.10.6.2 Pregnancy
Subjects will be counseled to inform the investigator of any pregnancy that occurs during the
study. If a subject becomes pregnant during the study, study interventions that may put the fetus
at risk will be stopped immediately.
reported to the medical monitor and Vertex Global Patient Safety (GPS) within 24 hours of the
site’s knowledge using the Pregnancy Information Collection Form.
obtained. A separate ICF will be provided to explain these follow-up activities. Birth outcomes

Baseline value, unless specified otherwise, will be defined as the most recent non-missing
measurement (scheduled or unscheduled) collected during screening and before start of
mobilization.
For number of severe VOC events: Pre-treatment baseline is defined as the average annual
severe VOC events during the 2 years prior to enrollment. Baseline will be calculated using
records of all severe VOC events during the 2 years before signing of the ICF.
All severe VOC events that occur after CTX001 infusion until the end of study (Month 24) will
be captured as post-CTX001 severe VOC events. All pre- and post-CTX001 infusion severe
VOC events will be adjudicated by an EAC, and only severe VOC events related to the
underlying SCD and not to an acute intercurrent event, such as acute bleeding or infection, will
be included for evaluating the change from baseline in annualized rate of VOC.
Change (absolute change) from baseline will be calculated as Post-baseline value − Baseline
value.
Treatment-emergent (TE) Period will include the time from CTX001 infusion to last study
visit.
evaluated as observed.
12.3.2 Background Characteristics
12.3.2.1 Subject Disposition
The number and percentage of subjects in the following categories will be summarized as
appropriate:
• Enrolled Set
• Subjects who are mobilized
• Subjects who are dosed with CTX001
• Subjects who are dosed with CTX001 with at least 1 year of follow-up post-CTX001
infusion
• Completed study (24-month post-CTX001 follow-up)
• Prematurely discontinued the study and the reasons for discontinuation
12.3.2.2 Demographics and Baseline Characteristics
• Age, sex, race, ethnicity, weight, height, genotype
• Targeted medical history
• Results of screening imaging (cardiac) and selected laboratory tests

• Relative change from baseline in annualized duration of hospitalization for severe VOC will
be summarized.
• Proportion of subjects achieving HbF ≥20% for at least 3 months, starting 3 months after
CTX001 infusion at the time of analysis, with the exact 95% CI will be provided.
• Proportion of subjects achieving HbF ≥20% for at least 3 months, starting at the time of
CTX001 infusion at the time of analysis, with the exact 95% CI will be provided.
• HbF and Hb concentrations will be summarized as a continuous variable over time.
• Relative change in number of units of RBC transfused for SCD-related indications will be
summarized.
• Change in PROs will be summarized as a continuous variable over time.
• Proportion of alleles with intended genetic modification present in bone marrow cells will be
12.3.3.3 Analysis of Exploratory Endpoints
• Change in hemolytic index as measured by principal component analysis of 4 markers of
hemolysis (reticulocyte count, serum concentrations of AST, LDH, and total bilirubin) will
• Change in TRV will be summarized as a continuous variable over time.
• Change in proportion of circulating erythrocytes expressing F-cells will be summarized as a
• Change in inflammatory markers will be summarized as a continuous variable over time.
• Change in endothelial activation markers will be summarized as a continuous variable over
time.
12.3.4 Safety Analysis
AEs will be coded according to MedDRA.
Analysis based on Safety Analysis Set will include:
• Proportion of subjects with neutrophil engraftment within 42 days after CTX001 infusion
• AEs, laboratory values, and vital signs from signing of informed consent through Month 24
visit.
• Incidence of TRM within 100 days and 1 year post CTX001 infusion. TRM defined as death
The number and percentage of subjects with AEs and treatment-emergent AEs will be
summarized by severity and seriousness in a tabular fashion according to the following time
periods: ICF signed to start of mobilization, mobilization to start of busulfan conditioning,

busulfan conditioning to start of CTX001 infusion, and CTX001 infusion through 24 months of
post-infusion.
Time to neutrophil engraftment, defined as the first of 3 consecutive days with ANC ≥500/μL
from transplantation, will be analyzed by the Kaplan-Meier method. Engraftment failure is
defined as not achieving neutrophil engraftment by Day 42 post-CTX001 infusion or receipt of
backup stem cells. The number and percentage of subjects with engraftment failure will be
summarized.
Time to platelet engraftment, defined as first 3 consecutive days with platelet ≥20,000/μL
without a transfusion in the past 7 days will be assessed by the Kaplan-Meier method.
Laboratory abnormalities (values outside of normal ranges, and by Common Terminology
Criteria for Adverse Events [CTCAE] grade), will also be tabulated.
The number and percentage of subjects with TRM within 100 days and 1 year post CTX001
infusion will be summarized, where TRM is defined as death at least possibly related to the
transplant-related.
12.3.4.1 Adverse Events
AEs will be classified as: AEs occurring from ICF signed to start of mobilization, from
mobilization to start of busulfan conditioning, from busulfan conditioning to start of CTX001
infusion, and from CTX001 infusion through 24 months of post-infusion.
AE summaries will be presented by MedDRA System Organ Class and Preferred Term using
frequency counts and percentages (i.e., number and percentage of subjects with an event). When
summarizing the number and percentage of subjects with an event, subjects with multiple
occurrences of the same AE or a continuing AE will be counted once. Only the maximum
severity level will be presented in the severity summaries, and the strongest relationship level
will be presented in the relationship summaries.
• AEs by strongest relationship
• AEs by maximum severity
• SAEs
• AEs leading to death
Details for imputing missing or partial start dates of AEs will be specified in the SAP.
12.3.4.2 Clinical Laboratory Assessments
hematology and chemistry results, including coagulation studies will be summarized in SI units
by visit.

12.3.4.3 Electrocardiogram
will be provided by visit for the following standard 12-lead ECG measurements: RR (msec), HR
HR intervals (QTcF [msec]).
12.3.4.4 Vital Signs
12.3.4.5 Physical Examination
PE findings will be presented in individual subject data listings only.
12.3.5 Other Analysis
Descriptive statistics will be used to describe the following parameters (unless stated otherwise):
• Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) from
baseline (pre-transfusion)
• CD34+ counts
• Correlations of response markers (transfusions, total and fetal hemoglobin) with
pre-treatment variables (e.g., α/non-α globin ratio, subject genotype), cell dose and percent
edited cells in final product
12.3.6 Interim and Independent Data Monitoring Committee Analyses
12.3.6.1 Interim Analysis
12.3.6.2 Independent Data Monitoring Committee Analysis

The DMC review will include review of safety and efficacy data. Details will be included in the
DMC charter and DMC SAP.

13 PROCEDURAL, ETHICAL, REGULATORY, AND ADMINISTRATIVE

CONSIDERATIONS
13.1 Adverse Event and Serious Adverse Event Documentation, Severity

Grading, and Reporting
13.1.1.1 Definition of an Adverse Event
frequency) after the ICF is signed. A pre-existing condition that is diagnosed prior to subject
signing ICF should be recorded in medical history.
An AE is considered serious if it meets the definition in Section 13.1.2.1.
13.1.1.2 Clinically Significant Assessments
Study assessments including laboratory tests, ECGs, PEs, and vital signs will be assessed and
those deemed to have clinically significant worsening from baseline will be documented as an
AE. When possible, a clinical diagnosis for the study assessment will be provided, rather than the
abnormal test result alone (e.g., urinary tract infection, anemia). In the absence of a diagnosis,
the abnormal study assessment itself will be listed as the AE (e.g., bacteria in urine or decreased
hemoglobin).
the following:
• Concomitant signs or symptoms related to the abnormal study assessment
• Further diagnostic testing or medical/surgical intervention
• Discontinuation from the study
A laboratory value that is Grade 4 will not automatically be an SAE. A Grade 4 laboratory value
will be an SAE if the subject’s clinical status indicates a life-threatening AE.
context, Grade 1 and 2 laboratory abnormalities reflective of and fully explained by busulfan
13.1.1.3 Documentation of Adverse Events
AEs will be monitored and collected as outlined in Table 13-1.

Table 13-1 AE and SAE Recording

mobilization cycle
Month 24 (End of Study Visit)
• Description of the event
• Classification of “serious” or “nonserious”
• Date of first occurrence and date of resolution (if applicable)
• Severity
• Causal relationship to study drug(s)
• Action taken
• Outcome
• Concomitant medication or other treatment given
13.1.1.4 Adverse Event Severity
guidance available at the following website will be consulted: CTCAE, Version 4.0, Cancer
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm (Accessed
November 2017). AEs of CTCAE Grades 4 and 5 will be documented as “life-threatening.” The
severity of an AE that does not appear in the CTCAE will be determined according to the
definitions in Table 13-2.

Table 13-2 Grading of AE Severity

Mild (Grade 1) Mild level of discomfort and does not interfere with regular activities
Moderate (Grade 2) Moderate level of discomfort and significantly interferes with regular activities
Severe (Grade 3) Significant level of discomfort and prevents regular activities
Life-threatening (Grade 4) Any adverse drug event that places the subject, in the view of the investigator, at
immediate risk of death
13.1.1.5 Adverse Event Causality

study drug(s). Causality will be classified using the categories in Table 13-3.
Table 13-3 Classifications for AE Causality

Related There is an association between the event and the administration of investigational
study drug, a plausible mechanism for the event to be related to the investigational
study drug and causes other than the investigational study drug have been ruled out,
and/or the event reappeared on re-exposure to the investigational study drug.
investigational study drug and there is a plausible mechanism for the event to be
related to investigational study drug, but there may also be alternative etiology, such
as characteristics of the subject’s clinical status or underlying disease.
Unlikely related The event is unlikely to be related to the investigational study drug and likely to be
related to factors other than investigational study drug.
Not related The event is related to an etiology other than the investigational study drug (the
alternative etiology will be documented in the subject’s medical record).
13.1.1.6 Study Drug Action Taken

The investigator will classify the study drug action taken with regard to the AE. The action taken
will be classified according to the categories in Table 13-4.
Table 13-4 Classifications for Study Drug Action Taken With Regard to an AE
Dose not changed Study drug dose not changed in response to an AE
Dose reduced Study drug dose reduced in response to an AE
Drug interrupted Study drug administration interrupted in response to an AE
Drug withdrawn Study drug administration permanently discontinued in response to an AE
Not applicable Action taken regarding study drug administration does not apply.
“Not applicable” will be used in circumstances such as when the investigational
treatment had been completed before the AE began and no opportunity to decide
whether to continue, interrupt, or withdraw treatment is possible.

13.1.1.7 Adverse Event Outcome

The outcome will be classified according to the categories in Table 13-5.
Table 13-5 Classifications for Outcome of an AE

Recovered/resolved Resolution of an AE with no residual signs or symptoms
Recovered/resolved with Resolution of an AE with residual signs or symptoms
sequelae
Not recovered/not Either incomplete improvement or no improvement of an AE, such that it remains
resolved (continuing) ongoing
Fatal Outcome of an AE is death. “Fatal” will be used when death is at least possibly
related to the AE.
Unknown Outcome of an AE is not known (e.g., a subject lost to follow up)
13.1.1.8 Treatment Given

The investigator ensures adequate medical care is provided to subjects for any AEs, including
clinically significant laboratory values related to study drug. In addition, the investigator will
describe whether any treatment was given for the AE. “Yes” is used if any treatment was given
in response to an AE, and may include treatments such as other medications, surgery, or physical
therapy. “No” indicates the absence of any kind of treatment for an AE.
13.1.2 Serious Adverse Events
13.1.2.1 Definition of a Serious Adverse Event
• Fatal (death, regardless of cause, that occurs during participation in the study or occurs after
participation and is suspected of being a delayed toxicity due to administration of the study
drug)
• Life-threatening, such that the subject was at immediate risk of death from the reaction as it
occurred
mobilization/apheresis, central line placement, busulfan conditioning, CTX001 infusion) do
not meet this criterion.
• Persistent or significant disability/incapacity (disability is defined as a substantial disruption
of a person’s ability to conduct normal life functions)
• Important medical event that, based upon appropriate medical judgment, may jeopardize the
subject or may require medical or surgical intervention to prevent 1 of the outcomes listed
above (e.g., an allergic bronchospasm requiring intensive treatment in an emergency room or
at home)

• Engraftment failure: failure to reach ANC ≥500 cells/µL on 3 consecutive days by Day 42
after CTX001 infusion or need to receive backup stem cells at any time during period of
neutropenia
• Development of new malignancy
“serious”, which is based on subject/event outcome or action described above, and is usually
associated with events that pose a threat to a subject’s life or functioning. Seriousness, not
13.1.2.2 Reporting and Documentation of Serious Adverse Events
24-Month Visit, regardless of causality, will be reported by the investigator to Vertex GPS
within 24 hours of identification. In addition, all SAEs that occur after the 24-Month Visit and
are considered related to study drug(s) will be reported to Vertex GPS within 24 hours of
identification.
Fax: +1-617-341-6159

13.1.2.3 Expedited Reporting and Investigator Safety Letters

The sponsor is responsible for reporting suspected, unexpected, serious adverse reactions
(SUSARs) involving the study drug(s) to all regulatory authorities, IECs, and participating
investigators in accordance with ICH Guidelines and/or local regulatory requirements, as
applicable. In addition, the sponsor, or authorized designee, will be responsible for the
submission of safety letters to central IECs.
It is the responsibility of the investigator or designee to promptly notify the local IRB/IEC of all
unexpected serious adverse drug reactions involving risk to human subjects. Investigators will
also be notified of all unexpected, serious, drug-related events (7/15 Day Safety Reports) that
occur during the study. Each site is responsible for notifying its IRB or EC of these additional
SAEs.
13.2 Administrative Requirements
13.2.1 Ethical Considerations
The study will be conducted in accordance with the current ICH GCP Guidelines, which are
consistent with the ethical principles founded in the Declaration of Helsinki, and in accordance
with local applicable laws and regulations. The IRB/IEC will review all appropriate study
documentation to safeguard the rights, safety, and well-being of the subjects. The study will be
conducted only at sites where IRB/IEC approval has been obtained. The protocol, Investigator’s
Brochure, sample ICF, advertisements (if applicable), written information given to the subjects
(including diary cards), safety updates, annual progress reports, and any revisions to these
documents will be provided to the IRB/IEC by the investigator or Sponsor, as allowable by local
applicable laws and regulations.
Ethics Committee/Institutional Review Board
Ethics Review
subjects.

13.2.2 Subject Information and Informed Consent

After the study has been fully explained, written informed consent will be obtained from the
subject or legal representative or guardian (if applicable) before study participation. The method
of obtaining and documenting the informed consent and the contents of the consent will comply
with ICH GCP and all applicable laws and regulations and will be subject to approval by the
sponsor or its designee.
13.2.3 Investigator Compliance
No modifications to the protocol will be made without the approval of both the investigator and
the Sponsor. Changes that significantly affect the safety of the subjects, the scope of the
investigation, or the scientific quality of the study (i.e., efficacy assessments) will require
IRB/IEC notification before implementation, except where the modification is necessary to
eliminate an apparent immediate hazard to human subjects. The Sponsor will submit all protocol
modifications to the required regulatory authorities.
When circumstances require an immediate departure from procedures set forth in the protocol,
the investigator will contact the Sponsor to discuss the planned course of action. If possible,
contact will be made before the implementation of any changes. Any departures from the
protocol will be fully documented in the source documentation and in a protocol deviation log.
13.2.4 Access to Records
The investigator will make the office and/or hospital records of subjects enrolled in this study
available for inspection by the Sponsor or its representative at the time of each monitoring visit
and for audits. The records will also be available for direct inspection, verification, and copying,
as required by applicable laws and regulations, by officials of the regulatory health authorities
(FDA and others). The investigator will comply with applicable privacy and security laws for use
and disclosure of information related to the research set forth in this protocol.
13.2.5 Subject Privacy
laws and regulations, all data provided to the Sponsor, study reports, and communications
relating to the study will identify subjects by assigned subject numbers, and access to subject
names linked to such numbers will be limited to the site and the study physician and will not be
disclosed to the Sponsor. As required by applicable laws and regulations in the countries in
which the study is being conducted, the investigator will allow Sponsor and/or its representatives
access to all pertinent medical records to allow for the verification of data gathered and the
review of the data collection process. The FDA and regulatory authorities in other jurisdictions,
including the IRB/IEC, may also request access to all study records, including source
documentation, for inspection.
For sites participating in the US, and in accordance with the Health Insurance Portability and
Accountability Act (HIPAA) and associated regulations, an executed HIPAA authorization will
be obtained by the site from each subject (or the legal representative of the subject) before
research activities may begin. Each HIPAA authorization will comply with all HIPAA
requirements including authorization allowing the site access to and use of the subject’s
personally identifiable health information, authorization for the site to disclose such information
to Sponsor, the FDA, and other parties requiring access under the protocol, and statements as to
the purpose for which such information may be used and for how long.

13.2.6 Record Retention

The investigator will maintain all study records according to ICH GCP Guidelines and/or
applicable local regulatory requirement(s), whichever is longest, as described in the Clinical
Trial Agreement. If the investigator withdraws from the responsibility of keeping the study
records, custody will be transferred to a person willing to accept the responsibility and the
Sponsor will be notified.
13.2.7 Study Termination
to:
• Subject or investigator noncompliance
• Unsatisfactory subject enrollment
• Lack of adherence to protocol procedures
• Lack of evaluable and/or complete data
• Potentially unacceptable risk to study subjects
• Decision to modify drug development plan
• Decision by the FDA or other regulatory authority
on a long-term follow-up study.
13.2.8 End of Study
End of study will be defined as the date the last subject completes the 24-month follow-up period
or date of early withdrawal from the study. The Sponsor will notify all applicable regulatory
agencies in accordance with local requirements when the study has ended.
13.3 Data Quality Assurance
Sponsor or its designated representative will conduct a study site visit to verify the qualifications
of each investigator, inspect clinical study site facilities, and inform the investigator of
responsibilities and procedures for ensuring adequate and correct study documentation.

The investigator is required to prepare and maintain adequate and accurate case histories
designed to record all observations and other data pertinent to the study for each subject. Study
data for each enrolled subject will be entered into a CRF by study site personnel using a secure,
validated, web-based electronic data capture (EDC) application. The Sponsor will have read-only
access to site-entered clinical data in the EDC application.
Instances of missing, discrepant, or uninterpretable data will be queried with the investigator for
resolution. Any changes to study data will be made to the CRF and documented in an audit trail,
which will be maintained within the clinical database.
13.4 Monitoring
• Determine the adequacy of the facilities
• Discuss with the investigator(s) and other personnel their responsibilities with regard to
comply with GCP Guidelines. On-site checking of the CRFs/SAE Forms for completeness and
clarity, cross-checking with source documents, and clarification of administrative matters will be
performed.
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal
• Provide information and support to the investigator(s)
• Confirm that facilities remain acceptable
• Confirm that the investigational team is adhering to the protocol, that data is being
are being performed
13.5 Electronic Data Capture
A CRF will be completed for each consented study subject. It is the investigator’s responsibility
to ensure the accuracy, completeness, clarity, and timeliness of the data reported in the subject’s
CRF. Source documentation supporting the CRF data will indicate the subject’s participation in

and subject status.
The audit trail entry will show the user’s identification information and the date and time of any
including any changes made to them, to endorse the final submitted data for the subjects for
CRF in the form of a compact disc (CD) or other electronic media will be placed in the
investigator’s study file.
13.6 Publications and Clinical Study Report
13.6.1 Publication of Study Results
information to any third party except to such of the investigator’s employees and staff as have
if the parties decide to proceed with it, for the purpose of conducting the study.
partners and associates, the FDA, and other government agencies. The investigator also
understands that, to allow for the use of the information derived from the study, the investigator
has the obligation to provide the Sponsor with complete test results and all data developed in the
study.
conditions of a separate written agreement between the Sponsor and the investigator and/or the
investigator’s institution.
13.6.2 Clinical Study Report
A clinical study report, written in accordance with the ICH E3 Guideline, will be submitted in
accordance with local regulations.

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15.2 Investigator Signature Page

Protocol #: CTX001-121 Version #: 1.0 Version Date: 13 April
2018
Study Title: A Phase 1/2 Study to Evaluate the Safety and Efficacy of a Single Dose of
Autologous CRISPR-Cas9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells
(CTX001) in Subjects With Severe Sickle Cell Disease
I have read Protocol CTX001-121, Version 1.0, and agree to conduct the study according to its
terms. I understand that all information concerning CTX001 and this protocol supplied to me is
confidential.
Printed Name
Signature Date

16 APPENDICES
16.1 Busulfan PK Collection

Recommendations for dosing q6h: Samples for busulfan PK analysis should be collected at the
above.
adjustments will be included in the electronic case report form (eCRF).

Page 1 of 91
1 TITLE PAGE
Clinical Study Protocol

A Phase 1/2 Study to Evaluate the Safety and
Efficacy of a Single Dose of Autologous
CRISPR-Cas9 Modified CD34+ Human Hematopoietic
Stem and Progenitor Cells (CTX001) in Subjects With
Severe Sickle Cell Disease
Study Number: CTX001-121
EudraCT Number: 2018-001320-19
Date of Protocol: 04 February 2020 (Version 5.0)
Vertex Pharmaceuticals Incorporated

50 Northern Avenue
Boston, MA 02210, USA
CONFIDENTIAL
This document contains confidential information. Any use, distribution, or disclosure without the prior written consent
of Vertex Pharmaceuticals Incorporated is strictly prohibited except to the extent required under applicable laws or
regulations. Persons to whom the information is disclosed must be informed that the information is confidential and
may not be further disclosed by them.
Summary of Changes to the Protocol

The previous version of this protocol (Version 4.0, 19 September 2019) was amended to create
the current version (Version 5.0, 04 February 2020). The protocol history is below:
Protocol History
Version and Date of Protocol Comments
Version 1.0, 13 April 2018 Original version
Version 2.0, 26 June 2018 Global version
Version 3.0, 10 December 2018 Global version (except Germany)
Version 2.1 [DE], 15 February 2019 German-specific version
Version 4.0, 19 September 2019 Global version (except Germany)
Version 5.0, 04 February 2020 Current global version (except Germany)
Key changes made in the current version of the protocol are summarized below.
Added expansion of the study to include subjects 12 to <18 years of age after Section 2, Section 8.1,
DMC review of of engraftment, efficacy and safety data from Section 9.1, Section 9.2,
at least subjects ≥18 years old. Section 9.6.2
Added that approximately adult subjects will be dosed with CTX001 before Section 2, Section 9.1,
the conditioning and dosing of the first pediatric subject. Section 9.2
Clarified that the study may be expanded to up to 45 subjects dosed. Section 2, Section 9.1,
Section 9.2, Section 12.1,
Section 12.3.8.1
For busulfan dosing, specified that for subjects less than 34 kg, institutional Section 2, Table 3-3, Section
and/or regional practice characteristics may be used. 9.1.3.1, Section 9.8.3
Added HbS and Hb collection for Days 1, 2, and 3 of Mobilization cycles 1, 2, Table 3-2
and 3 in schedule of assessments to align with text in Section 11.2
Added that Hb concentration will also be documented after transfusion to be Section 11.2
consistent with HbS collection.
Added the option for parents to sign assent for pediatric subjects and updated Section 2, Section 8.1,
related text in the protocol body Table 3-1 through Table 3-4,
Section 9.7.1, Section 9.10,
Section 11.9, Section 13.2.2
Added transcranial Doppler (TCD) ultrasonography for pediatric subjects 12 to Table 3-1, Section 8.1,
16 years old Section 11.4
Added history of abnormal TCD (TAMMV ≥200 cm/sec) for subjects 12 to 18 Section 8.2
years of age as an exclusion criterion
As the Karnofsky performance status is used only for subjects ≥16◦years old, the Section 8.1, Section 11.12.3
Lansky performance status scale was added for use in subjects <16◦years old.
Updated the inclusion criteria and contraception requirements to specify that Section 8.1, Section 11.12.6.1
they apply to subjects of reproductive capacity and who are sexually active;
added that subjects whose contraception waiver status changes during their
participation in the study will be required to follow the contraception
requirements.
Added youth and parent proxy versions of EQ-5D for subjects <18 years old Section 2, Table 3-1,
Table 3-4, Section 7.4,
Section 11.9.2
Vertex Pharmaceuticals Incorporated Confidential Information


Added Wechsler Abbreviated Scale of Intelligence for subjects 12 to <18 years Table 3-2, Section 11.10,
old at the time of assessment because there is not a validated youth version of Section 11.11
the Mini-Mental State Examination. Specified that the Mini-mental State
Examination is for adults.
Added Pediatric Quality of Life Inventory (teen and parent proxy versions) Section 2, Table 3-1,
assessment because a pediatric version of the FACT-BMT does not exist Table 3-4, Section 7.4,
Section 11.9.5
Added PedsQL Sickle Cell Disease Module (teen and parent proxy versions) for Section 2, Table 3-1,
subjects 12 to <18 years of age Table 3-4, Section 7.4,
because ASCQ-Me is not suitable for these subjects Section 11.9.5
Provided therapeutic rationale for inclusion of pediatric subjects Section 9.6.1, Section 9.6.2
Included statement on risk of study procedures for pediatric subjects Section 9.6.1
Included description of therapies to prevent complications of SCD Section 9.6.2
Updated enrollment suspension criteria and individual stopping rules to include Section 9.6.1, Section 9.9.1,
failure to manufacture the target cell dose of CTX001 instead of failure to Section 9.9.2
collect the target number of CD34+ cells.
Clarified that subjects will be removed from the study if they have an important Section 9.9.2
protocol deviation prior to the start of busulfan conditioning that, in the opinion
of the investigator, would put the subject at risk for continuing study-related
procedures or follow-up.
Added the option for gonadal tissue banking for pre-pubescent subjects, if Section 2, Table 3-2,
appropriate per subject age and local site practice Section 11.8
Removed the phrase “or by demonstration of the absence of the vas deferens by Section 11.12.6.1
ultrasound” for documentation of male infertility.
Added that AEs collection will exclude AEs possibly related to fertility Table 13-1
preservation procedures
Clarified for subjects that went through screening and rescreening, any VOCs Section 12.3.1
that occurred during the initial screening(s) and the last screening will be
counted toward the baseline calculation.
Removed redundant exploratory endpoint: "Change in proportion of circulating Section 12.3.7
erythrocytes expressing F-cells will be summarized as a continuous variable
over time."
Clarified timing for sample collection for busulfan PK collection Appendix 16.1
Specified timeframe for the observation of Grade 3 or greater AEs that might Section 2, Section 9.1,
trigger a DMC. Section 9.2
In exclusion criterion 14, the upper limit for direct bilirubin was changed from Section 8.2
2 × ULN to 2.5 × ULN. The original limit was more stringent than required,
because the criterion also assesses other measures that are collectively more
robust assessments of advanced liver disease than direct bilirubin alone.
Clarified that historical VOCs that occur within the 2-year period, including Section 9.4
those which may begin just prior to the 2-year window and end during the
2-year window, will contribute to the determination of eligibility
Section 12.3.3.1
Added that subgroup analyses, such as descriptive summaries of the key efficacy
/ safety endpoints by age groups as well as by genotypes, will be provided as
appropriate. Details will be described in the SAP.
Typographical and administrative changes were also made to improve the clarity of the
document.

2 PROTOCOL SYNOPSIS
Title A Phase 1/2 Study to Evaluate the Safety and Efficacy of a Single Dose of
Autologous CRISPR-Cas9 Modified CD34+ Human Hematopoietic Stem and
Progenitor Cells (CTX001) in Subjects With Severe Sickle Cell Disease
Brief Title A Study to Evaluate the Safety and Efficacy of a Single Dose of CTX001 in
Subjects With Severe Sickle Cell Disease
Clinical Phase and Phase 1/2 Safety and Efficacy Study

Clinical Study Type
Objectives Primary Objective

Evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9
modified CD34+ human hematopoietic stem and progenitor cells (hHSPCs)
(CTX001) in subjects with severe sickle cell disease (SCD)
Secondary Objectives
 Assess the effects of infusion of CTX001 on disease-specific events and
clinical status
 Quantify gene editing efficiency
Exploratory Objective
Assess the ability of biomarkers to characterize CTX001 effect and predict
treatment outcomes
Endpoints Primary Endpoints

Safety Endpoints
 Successful neutrophil engraftment within 42 days after CTX001 infusion
 Time to neutrophil engraftment
 Safety and tolerability assessments based on adverse events (AEs), clinical
laboratory values, and vital signs
 Transplant-related mortality (TRM) within 100 days after CTX001 infusion
 TRM within 1 year after CTX001 infusion
 All-cause mortality
 Proportion of subjects with sustained fetal hemoglobin (HbF) ≥20% at the
time of analysis for at least 3 months starting 6 months after CTX001
infusion, in the absence of treatment with hydroxyurea (HU)
 Relative change from baseline in annualized rate of severe vaso-occlusive
crises (VOCs), starting 6 months after CTX001 infusion
Secondary Endpoints
 Reduction in annualized rate of severe VOCs at the time of analysis from
baseline by at least 50%, starting 6 months after CTX001 infusion
 Reduction in annualized rate of severe VOCs at the time of analysis from
baseline by at least 65%, starting 6 months after CTX001 infusion

 Absence of severe VOCs for at least 12 months at the time of the analysis,
 Relative change from baseline in annualized duration of hospitalization for
severe VOCs, starting 6 months after CTX001 infusion
 Relative change from baseline in annualized rate of hospitalization for severe
VOCs starting 6 months after CTX001 infusion
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for at
least 3 months, starting 3 months after CTX001 infusion, in the absence of
treatment with HU
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for at
least 3 months, starting at the time of CTX001 infusion, in the absence of
treatment with HU
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for
6 months, starting 6 months after CTX001 infusion, in the absence of
treatment with HU
 Change in number of units of red blood cells (RBC) transfused for
SCD-related indications over time
 HbF concentrations over time
 Hemoglobin (Hb) concentrations over time
 Proportion of alleles with intended genetic modification present in peripheral
blood leukocytes over time
 Proportion of alleles with intended genetic modification present in bone
marrow cells over time
 Change in patient reported outcomes (PROs) over time in adults (≥18 years)
using;
o Pain-scale:11-point numerical rating scale (NRS)
o Functional assessment of cancer therapy-bone marrow transplant
(FACT-BMT)
o Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me)
o EuroQol Quality of Life Scale (EQ-5D-5L)
 Change in PROs over time in adolescents (12 to <18 years of age) using;
o Pain-scale: 11-point NRS
o Pediatric Quality of Life Inventory (PedsQL Teen and parent proxy
versions)
o PedsQL SCD module (Teen and parent proxy versions)
o EQ-5D-Youth (EQ-5D-Y Self-report and parent proxy version)
 Change in hemolytic index as measured by principal component analysis of
4 markers of hemolysis (reticulocyte count, serum concentrations of aspartate
transaminase, lactate dehydrogenase [LDH], and total bilirubin) over time
 Change in tricuspid regurgitant jet velocity (TRV) over time
 Change in proportion of circulating erythrocytes expressing fetal hemoglobin
(F-cells) over time
 Change in inflammatory and endothelial activation markers over time
Number of Subjects Up to 17 subjects with possible expansion to a total of up to 45 subjects dosed

Study Population Subjects 12 to 35 years of age (inclusive at time of informed consent) with
documented βS/βS or βS/β0 genotype who have severe SCD. Severe SCD is
defined by the occurrence of at least 2 of the following events each year during
the 2-year period before screening, while receiving appropriate supportive care
(e.g., pain management plan, HU if indicated)
 Acute pain event that requires a visit to a medical facility and administration
of pain medications (opioids or intravenous [IV] non-steroidal
anti-inflammatory drugs [NSAIDs]) or RBC transfusions
 Acute chest syndrome, as indicated by the presence of a new pulmonary
infiltrate associated with pneumonia-like symptoms, pain, or fever
 Priapism lasting >2 hours and requiring a visit to a medical facility
 Splenic sequestration, as defined by an enlarged spleen, left upper quadrant
pain, and an acute decrease in hemoglobin concentration of ≥2 g/dL.
Investigational Drug Drug product: CTX001

Description: CTX001 is composed of autologous CD34+ hHSPCs modified with
CRISPR-Cas9 at the erythroid lineage-specific enhancer of the BCL11A gene.
Route of administration: IV infusion
Study Design This is a single-arm, open-label, multi-site, single-dose, Phase 1/2 study in
subjects with severe SCD. The study will evaluate the safety and efficacy of a
single dose of autologous CRISPR-Cas9 modified hHSPCs (CTX001) and will
include up to 17 subjects, 12 to 35 years of age, inclusive. The study may be
expanded to include a total of up to 45 subjects dosed.
Stage 1: Screening and Pre-mobilization Period
During this period, subjects who meet the inclusion criteria have the option to
undergo fertility preservation via cryopreservation of oocyte or sperm. For pre-
pubescent subjects, gonadal tissue collection and cryopreservation may be
performed per local practice and standard of care. After eligibility is confirmed,
subjects will begin RBC exchange transfusions for a minimum of 8 weeks before
the planned start of mobilization and will continue receiving these transfusions
until they begin busulfan conditioning. The goal of these RBC transfusions is to
maintain hemoglobin S (HbS) level of <30% of total Hb while keeping total Hb
concentration ≤11 g/dL. Treatment with HU should be stopped at least 8 weeks
before planned mobilization.
Subjects should receive an RBC exchange transfusion 3 (± 1) days before the start
of each mobilization/apheresis cycle.
Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection, CTX001
Manufacture and Disposition
Each subject will undergo stem cell mobilization with plerixafor only. Peripheral
blood mononuclear cells (PBMC) will be collected by apheresis.
On Day 1, subject will receive plerixafor .
Day 2 will be the same as Day 1. Day 3 (if required after Cycle 1) will be used to
collect an additional 2 × 106 CD34+ cells/kg as a back-up for rescue therapy in the
event of non-engraftment with CTX001. The targeted CD34+ cell collection is at
least 15 × 106 CD34+ cells/kg for manufacturing of CTX001 in order to achieve a
minimum target dose of 3 × 106 CD34+ cells/kg.

If the first mobilization and apheresis cycle does not yield enough cells for both
the minimum CTX001 product and safety backup or if a subject cannot complete
apheresis, up to 2 additional mobilization and apheresis cycles will be allowed to
collect additional cells. The additional mobilization cycle will be initiated at least
14 days after the first day of the prior mobilization cycle.
Stage 3: Myeloablative Conditioning (Stage 3A) and Infusion of CTX001 (Day 1,
Stage 3B)
Stage 3A – Myeloablative Conditioning: During CTX001 manufacturing and
before the planned start of busulfan conditioning, subjects will continue to receive
RBC exchange transfusions with the goal of maintaining an HbS level of <30% of
total Hb while keeping total Hb concentration ≤11 g/dL.
If the planned start of busulfan conditioning is >4 months after completion of
mobilization, the investigator may stop the RBC transfusion regimen and restart
HU for those subjects who have been previously treated with HU. If the RBC
transfusion regimen is interrupted, subjects should begin RBC exchange
transfusions a minimum of 8 weeks before the planned start of busulfan
conditioning with the goal of maintaining an HbS level of <30% of total Hb while
keeping total Hb concentration ≤11 g/dL. Subjects should receive an RBC
exchange transfusion 3 (± 1) days before the start of busulfan conditioning.
After the CTX001 product is received at the site and it has been confirmed that the
backup cells remain available and in acceptable condition to be administered if
needed, the subject will receive busulfan. The starting dose of busulfan will be
3.2 mg/kg administered IV once daily for 4 consecutive days. However, busulfan
may be administered as 0.8 mg/kg every 6 hours (q6h) for 4 consecutive days, per
the site’s standard practice. For subjects less than 34 kg, institutional and/or
regional practice may be used.
Stage 3B – CTX001 Infusion:
Infusion of CTX001 will occur at a minimum of 48 hours after the completion of
the busulfan infusion and at a maximum of 7 days after the completion of the
busulfan infusion.
On Day 1, the entire dose (all vial[s]) of CTX001 will be thawed and administered
through a central venous catheter.
Stage 4: Follow-up Through Engraftment and Up To 2 Years After CTX001
infusion
Stage 4A – Post-infusion In-hospital Follow-up: Subjects will be monitored daily
in the transplant unit and receive supportive care according to standard practices
for subjects undergoing hematopoietic stem cell transplant (HSCT).
Stage 4B – Post-engraftment Follow-up: After discharge from the transplant unit
subjects will be followed for approximately 2 years after the CTX001 infusion.
Study Duration The duration of Stage 1 will be approximately 2 to 4 months; Stage 2

approximately 2 to 4 months; Stage 3 approximately 1 month; Stage 4
approximately 2 years.
Subjects will be followed on study for a total of approximately 2 years after
CTX001 infusion.
Additionally, all subjects who receive CTX001 will be asked to enroll in the long-
term follow-up study (Study VX18-CTX001-131), after completion or
withdrawal/discontinuation from Study CTX001-121.
Assessments Efficacy: HbF levels, VOCs, RBC transfusions, alleles with intended genetic
modification, PROs

3 SCHEDULE OF ASSESSMENTS
Schedules of assessments are in Table 3-1, Table 3-2, Table 3-3, Table 3-4, and Table 3-5.

Screening
Informed consent and where applicable, X Perform vital signs after consent even if
assent collected prior to consent as part of standard
of care
PRO assessment (≥18 years old): X Perform as the first assessment after signing
Pain scale (11-point NRS), ASCQ-Me, informed consent (Section 11.9).
FACT-BMT, and EQ-5D-5L ASCQ-Me will include the following
7 questionnaires:
ASCQ-Me SCD Medical History Checklist;
PRO assessment (<18 years old): ASCQ-Me V 2.0_Sleep Impact Short Form;
Pain scale (11-point NRS), EQ-5D-Y, ASCQ-Me Short Form 2.0_Pain Impact;
PedsQL, and PedsQL SCD module ASCQ-Me SCD Pain Episode Frequency
Severity; ASCQ-Me Short Form 2.0_Social
Functioning Impact; ASCQ-Me Short Form
2.0_Emotional Impact; ASCQ-Me Short
Form 2.0_Stiffness Impact
Demographics X Section 11.1
Medical history X Including medical records on SCD-related
complications and associated treatment
including medical facility visits, inpatient
hospitalizations and transfusion records for at
least 2 years before consent will be collected
Full physical examination X Including assessment of spleen size
(Section 11.12.3)
Performance status X Section 11.12.3
Height and weight X Measure with shoes off
Vital signs X Includes blood pressure (systolic and
diastolic), temperature, pulse rate, respiration
rate, and pulse oximetry (Section 11.12.3)
SCD genotyping (central laboratory) X All subjects will be genotyped for SCD
(alpha and HBB loci) to confirm βS/βS or
βS/β0 genotype. Results will be required
before busulfan conditioning begins.
Serum chemistry X Section 11.12.2
Coagulation X Section 11.12.2
Hematology X Section 11.12.2
Serum pregnancy test X For females of childbearing potential
(Section 11.12.6)
Hemoglobin fractionation X HbF for establishing eligibility; will also
(central laboratory) include HbS, HbA2, HbA.
Infectious disease marker testing X Infectious disease marker testing will include
HBV (HBcAb, HbsAg, and NAT); HCV
(HCAb and NAT); HIV-1, HIV-2
(Antigen/Antibody immunoassay, if positive,
then NAT); and syphilisa HTLV-1 Ab (if
applicable) (Section 11.12.2)
Urinalysis X Section 11.12.2


Screening
12-lead ECG X Prior to any blood draws (Section 11.12.5)
Brain MRI/MRA X To confirm absence of active Moyamoya
disease. If a brain MRI/MRA has been
performed within 3 months before date of
consent, these results may be used, unless
repetition of the assessment is clinically
indicated (Section 11.4)
Transcranial Doppler (TCD) ultrasound X For subjects 12 to 16 years of age
(Section 11.4)
Echocardiograph X LVEF, TRV, and E/e’ assessment
(Section 11.4)
DLco [corrected] X Section 11.3
Adverse event assessment X Continuous from signing ICF and assent
(where applicable)
Prior and concomitant medications X Continuous from signing ICF and assent
(where applicable)
aSubjects must have at least a screening assay for syphilis performed (e.g., rapid plasma reagin [RPR] is preferred). If positive,
a confirmatory test (e.g., FTA-ABS, TPPA) must be done to confirm the presence of active infection. Local testing algorithms
for syphilis may be used, provided they contain these tests.

Mobilization and Apheresisb
After Eligibility
Confirmation
(Before
Mobilization Day -5 to Day 3
Event/Assessment Start) a,b Day -1a Day 1 Day 2 (if needed) Comments
Blood allelic editing (central X Peripheral blood sample; Collect before start of first RBC exchange transfusion
laboratory) and before first cycle only (Section 11.5)
Immunological testing X Collect before start of first RBC exchange transfusion, and repeated before
each cycle (Section 11.12.2)
HbF distribution, F-cells X Collect before start of first RBC exchange transfusion and before first cycle
(central laboratory) only (Section 11.5)
Inflammatory and endothelial X Collect before start of first RBC exchange transfusion and before first cycle
activation markers (central only (Section 11.5)
laboratory)
Bone marrow aspirate (central X Collect before start of first cycle only. Baseline sample for genomic analysis,
laboratory) and exploratory biomarkers (Section 11.7).
Exploratory biomarker samples X Collect before start of first RBC exchange transfusion and before first cycle
(central laboratory) only (Section 11.5.1)
Sperm or oocyte banking X (optional) If requested by subject or their legally authorized representative, if less than
18 years of age, prior to busulfan conditioning. Gonadal tissue banking may be
done as appropriate per subject age and local site practice. (Section 11.8)
X Repeated within 28 days before the start of each planned apheresis cycle and
Infectious disease marker
results should be reported at least 5 business days before each planned cycle
testing
(Section 11.12.2)
Mini-Mental State Examination Section 11.10, Section 11.11
(subjects ≥18 years old) or Age at time of assessment
Wechsler Abbreviated Scale of X
Intelligence (subjects <18 years
old)
X Collect before start of first RBC exchange transfusion and repeated before each
Hemolysis markers cycle. Hemolysis markers include haptoglobin and lactate dehydrogenase
(Section 11.12.2)
Females of childbearing potential only. Must be performed within 3 days prior
Serum pregnancy test X
to starting each mobilization.
Eligibility for Per site’s standard guidelines. Subject must be hemodynamically stable and
mobilization/apheresis X have no active infection per investigator judgment.
re-confirmed
Abbreviated physical Prior to each cycle and daily before starting each mobilization/apheresis day
X X X X
examination (Section 11.12.3)
Prior to each cycle and daily before starting each mobilization/apheresis day
Performance status X X X X
(Section 11.12.3)

Mobilization and Apheresisb
After Eligibility
Confirmation
(Before
Mobilization Day -5 to Day 3
Event/Assessment Start) a,b Day -1a Day 1 Day 2 (if needed) Comments
Adverse event assessment Continuous from signing ICF and assent (where applicable)
Prior and concomitant Continuous from signing ICF and assent (where applicable)
medications
a
Assessments may be performed over multiple days or visits
b
In addition to study-related visits, additional assessments or visits may be performed per institutional guidelines or as deemed appropriate by the investigator.
c
For subsequent cycles (after the first mobilization/apheresis cycle) Days 2 and 3 of apheresis will be performed as needed.

Pre-conditioninga,b Conditioningb Infusionb

30 to 7 Days 3 to 7 Days Before Day -5 to -2 Day 1
Before Busulfan Busulfan Initiation
Event/Assessment Initiation Comments
Serum pregnancy test X Females of childbearing potential only. Must be performed within
5 days of starting busulfan
Abbreviated physical X X X Section 11.12.3
examination
Performance status X X X Section 11.12.3
Vital signs X X X Includes blood pressure (systolic and diastolic), temperature,
pulse rate, respiration rate, and pulse oximetry.
Day 1: before infusion, and every 30 (± 5) minutes after start of
infusion and up to 2 hours post completion of infusion
(Section 11.12.3)
12-lead ECG X X Day 1: ECGs before start of infusion (Section 11.12.5)
Hematology X X X Day 1: Collect laboratory assessments before start of infusion
(Section 11.12.2)
Serum chemistry X X X Day 1: Collect laboratory assessments before start of infusion
(Section 11.12.2)
Eligibility for myeloablation X X 30 to 7 days before busulfan initiation: If screening and
reconfirmed pre-conditioning visits are ≥4 months apart, repeat pulmonary
function tests and echocardiograph
3 to 7 days before busulfan initiation: Review most recent
assessments (within 7 days prior to conditioning) as per site’s
standard guidelines for autologous stem cell transplant.
Pre-conditioning transfusion X Refer to Table 3-5. Treatment with HU should be stopped at least
regimen 8 weeks before planned mobilization.
Confirm rescue cells are X
cryopreserved in acceptable
condition at the site
Confirm CTX001 has been X
received in acceptable condition
at the site prior to initiating
busulfan
Test dose of busulfan to X (optional)
determine optimal busulfan
dosec
Anti-seizure prophylaxis X Initiate anti-seizure prophylaxis at least 12 hours before first dose
of busulfan per hospital guidelines
IV busulfan administration X Section 9.8.3

Pre-conditioninga,b Conditioningb Infusionb

30 to 7 Days 3 to 7 Days Before Day -5 to -2 Day 1
Before Busulfan Busulfan Initiation
Event/Assessment Initiation Comments
Busulfan PK d X Blood sample will be collected for PK analysis for busulfan on
X
Day 1 and Day 3 of conditioning as per Appendix 16.1.
IV infusion of CTX001 X Subjects should be pre-medicated with an antihistamine
(diphenhydramine hydrochloride) and an antipyretic
(acetaminophen or paracetamol) per institutional guidelines
(Section 10.1.4)
Adverse event assessment Continuous from signing ICF and assent (where applicable)
Prior and concomitant Continuous from signing ICF and assent (where applicable)
medications
a
b
In addition to study-related visits, subjects will be followed per institutional guidelines or as deemed appropriate by the investigator.
c For subjects less than 34 kg institutional or regional practice may be used
d PK analysis will be performed if the test dose of busulfan is administered

Daily Follow-upa,b
From D+12 D+15
D+30c D+60 D+90 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 0 0
Event/ Until M1 M2 M3 M4 M5 M6 M9 M12 M15 M18 M21 M24 ETFcd
Assessment Discharge (±4d) (±7d) (±7d) (±7d) (±7d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d) (±14d) Comments
Vital signs X X X X X X X X X X X X X X Includes blood pressure
(systolic and diastolic),
temperature, pulse rate,
respiration rate, and pulse
oximetry. (Section 11.12.3)
Abbreviated X X X X X X X X X X X X X X Section 11.12.3
physical
examination
Performance status X X X X X X X X X X X X X X Section 11.12.3
Post CTX001 X Refer to Table 3-5
infusion
transfusion
regimen
Hematology X X X X X X X X X X X X X X Section 11.12.2
Serum chemistry X X X X X X X X X X X X X X Section 11.12.2
Coagulation X X X X X X X X X X X X X X Section 11.12.2
X X X X X X X X X X Hemolysis markers include
haptoglobin and lactate
Hemolysis markers
dehydrogenase
(Section 11.12.2)
Immunological X X X X X Section 11.12.2
testing
Blood allelic X X X X X X X X X X X Before scheduled
editing (central transfusion (if applicable)
Hemoglobin X X X X X X X X X X X X X Before scheduled
fractionation transfusion (if applicable)
(central laboratory) (Section 11.5)
HbF distribution, X X X X X X X X X X X X X Before scheduled
F-cells (central transfusion (if applicable)
Inflammatory and X X X X X X Before scheduled
endothelial transfusion (if applicable)
activation markers (Section 11.5)

Daily Follow-upa,b
From D+12 D+15
D+30c D+60 D+90 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 0 0
Exploratory X X X X X X Before scheduled
biomarker blood transfusion (if applicable)
samples (central (Section 11.5.1)
laboratory)
Bone marrow X X X X For assessment of allelic
aspirate (central editing, and exploratory
laboratory) biomarkers (Section 11.7)
DLco [corrected] X X X Section 11.3
Echocardiograph X X X X X TRV, LVEF, and E/e’
assessment (Section 11.4)
12-lead ECG X X X Section 11.12.5

PRO assessments X X X X X X Assessment should be
For ≥18 years of performed as the first
age: assessment at any visit if
EQ-5D-5L, possible (Section 11.9)
FACT-BMT, and ASCQ-Me will include the
ASCQ-Me following 7 questionnaires:
ASCQ-Me SCD Medical
For <18 years of History Checklist; ASCQ-
age: Me V 2.0_Sleep Impact
EQ-5D-Y, Short Form; ASCQ-Me
PedsQL, and Short Form 2.0_Pain
PedsQL SCD Impact; ASCQ-Me SCD
module Pain Episode Frequency
Severity; ASCQ-Me Short
Form 2.0_Social
Functioning Impact; ASCQ-
Me Short Form
2.0_Emotional Impact;
ASCQ-Me Short Form
2.0_Stiffness Impact
PRO assessments X X X X X X X X X X X Assessment should be
(Pain-Scale performed as the first
[11-point NRS]) assessment at any visit if
possible (Section 11.9)
Adverse event Continuous from signing ICF and assent (where applicable)
collection

Daily Follow-upa,b
From D+12 D+15
D+30c D+60 D+90 D+180 D+270 D+360 D+450 D+540 D+630 D+720
Day 2 0 0
Prior and Continuous from signing ICF and assent (where applicable)
concomitant
medication
a
b
In addition to study-related visits, subjects will be followed per institutional guidelines or as deemed appropriate by the investigator.
c
Month (M) is defined as 30 days. If Day 30/M1 occurs while the subject is still hospitalized, the daily assessments required before discharge should be done in addition to the Day 30/M1
assessments.
d
Early Termination of Follow-Up (ETF). Assessments performed within 2 weeks of ETF Visit should not be repeated. Echocardiograph, DLco, and bone marrow aspirate should not be repeated if
performed within 6 months of ETF Visit.

Table 3-5 Study CTX001-121: Transfusion Regimen
Pre-mobilization Prior to Pre-conditioning After Start of
Mobilization Post CTX001
Busulfan to CTX001 Notes
Event/ and Apheresis Minimum 8 Infusion
Minimum 8 weeks 3 (±1) days Infusion
Assessment 3 (±1) days weeks
a Alltransfusions of RBC products should be
depleted of leukocytes and matched for at least the
ABO blood group, the Kell (K) blood group, and
Rh antigens C, D, and E
b Initiate RBC exchange transfusion a minimum of

8 weeks before planned start of mobilization with
a goal of maintaining the target HbS level of
<30% of total Hb while keeping total Hb
RBC exchange
Xb Xc Xd Xc None Xe c
transfusiona Subjects should receive an RBC exchange
transfusion
d Continue or reinitiate RBC exchange transfusion

a minimum of 8 weeks before start of busulfan
conditioning with a goal of maintaining an HbS
level of <30% of total Hb while keeping total Hb
e
As needed to maintain Hb ≥7 g/dL per
institutional standards.
Platelet
X Target to maintain platelets >50,000/L
transfusions
HbS and Hb
X X X
levels Collect prior to and after each transfusion

4 TABLE OF CONTENTS
1 Title Page ................................................................................................................................ 1

Summary of Changes to the Protocol......................................................................................... 2
2 Protocol Synopsis ................................................................................................................... 4
3 Schedule of Assessments........................................................................................................ 9
4 Table of Contents ................................................................................................................. 20
List of Tables............................................................................................................................ 23
List of Figures .......................................................................................................................... 24
List of Abbreviations................................................................................................................ 25
Glossary of Terms .................................................................................................................... 28
5 Introduction.......................................................................................................................... 29
5.1 Background..................................................................................................................... 29
5.1.1 Hemoglobinopathies ............................................................................................... 29
5.1.2 Sickle Cell Disease ................................................................................................. 29
5.2 Therapeutic Rationale ..................................................................................................... 30
5.2.1 CRISPR-Cas9 Gene Editing Approach .................................................................. 31
5.2.2 BCL11A Erythroid-lineage Specific Enhancer for Editing..................................... 33
5.2.3 CTX001 .................................................................................................................. 35
5.3 Nonclinical Experience .................................................................................................. 35
5.4 Clinical Experience ........................................................................................................ 35
6 Study Objectives .................................................................................................................. 36
6.1 Primary Objective ........................................................................................................... 36
6.2 Secondary Objectives ..................................................................................................... 36
6.3 Exploratory Objective .................................................................................................... 36
7 Study Endpoints ................................................................................................................... 36
7.1 Primary Endpoints .......................................................................................................... 36
7.2 Primary Efficacy Endpoint ............................................................................................. 36
7.2.1 Safety Endpoints ..................................................................................................... 36
7.3 Key Secondary Efficacy Endpoint ................................................................................. 36
7.4 Secondary Endpoints ...................................................................................................... 36
7.5 Exploratory Endpoints .................................................................................................... 37
8 Study Population.................................................................................................................. 38
8.1 Inclusion Criteria ............................................................................................................ 38
8.2 Exclusion Criteria ........................................................................................................... 39
9 Study Implementation ......................................................................................................... 40
9.1 Study Design .................................................................................................................. 40
9.1.1 Stage 1: Screening and Pre-mobilization Period .................................................... 41
9.1.1.1 Rescreening and Repetition of Screening Assessment(s) ................................ 42
9.1.2 Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection, CTX001
Manufacture and Disposition.................................................................................. 42
9.1.3 Stage 3: Myeloablative Conditioning (Stage 3A) and Infusion of CTX001 (Day 1,
Stage 3B) ................................................................................................................ 43
9.1.3.1 Stage 3A: Myeloablative Conditioning............................................................ 44
9.1.3.2 Stage 3B: CTX001 Infusion (Day 1) ............................................................... 44

9.1.4 Stage 4: Follow-up Through Engraftment and Up To 2 Years After CTX001

Infusion ................................................................................................................... 45
9.1.4.1 Stage 4A: Post-infusion In-hospital Follow-up (Engraftment) ........................ 45
9.1.4.2 Stage 4B: Post-engraftment Follow-up ............................................................ 45
9.2 Data Monitoring Committee........................................................................................... 45
9.3 Steering Committee ........................................................................................................ 46
9.4 Endpoint Adjudication Committee ................................................................................. 46
9.5 Method of Assigning Subjects to Treatment Groups ..................................................... 46
9.6 Rationale for Study Elements ......................................................................................... 46
9.6.1 Study Design........................................................................................................... 46
9.6.1.1 Primary Efficacy Endpoint ............................................................................... 47
9.6.2 Study Population..................................................................................................... 48
9.6.3 Dose ........................................................................................................................ 49
9.6.4 Study Duration ........................................................................................................ 50
9.7 Prior and Concomitant Treatments and Procedures ....................................................... 50
9.7.1 Prior Medications ................................................................................................... 50
9.7.2 Venous Access ........................................................................................................ 50
9.7.3 Prohibited Medications ........................................................................................... 50
9.8 Administration ................................................................................................................ 51
9.8.1 RBC Transfusion .................................................................................................... 51
9.8.2 Mobilization and Apheresis .................................................................................... 51
9.8.2.1 Mobilization ..................................................................................................... 51
9.8.2.2 Apheresis Procedure......................................................................................... 52
9.8.3 Conditioning: Busulfan Administration ................................................................. 52
9.8.4 CTX001 Infusion .................................................................................................... 53
9.8.5 Post-CTX001 Infusion Infection Prophylaxis and Surveillance ............................ 53
9.9 Enrollment Suspension Criteria and Stopping Rules ..................................................... 53
9.9.1 Enrollment Suspension Criteria .............................................................................. 53
9.9.2 Stopping Rules for Individual Subjects .................................................................. 55
9.10 Removal of Subjects ....................................................................................................... 55
9.11 Replacement of Subjects ................................................................................................ 56
10 Study Drug Information and Management ....................................................................... 56
10.1 Preparation and Dispensing ............................................................................................ 56
10.1.1 Manufacture of CTX001 ........................................................................................ 56
10.1.2 Shipment of CTX001 Product to Treatment Site ................................................... 56
10.1.3 Storage of CTX001 Product ................................................................................... 56
10.1.4 CTX001 Infusion Procedures, Dose, and Administration ...................................... 56
10.2 Drug Accountability ....................................................................................................... 57
10.3 Disposal of Unused Drug ............................................................................................... 57
10.4 Blinding and Unblinding ................................................................................................ 57
11 Assessments .......................................................................................................................... 57
11.1 Subject and Disease Characteristics ............................................................................... 58
11.2 Transfusions ................................................................................................................... 58
11.3 Diffusing Capacity of the Lungs for Carbon Monoxide ................................................ 58
11.4 Imaging Assessments ..................................................................................................... 58
11.5 Blood for Biomarker Assessments ................................................................................. 58

11.5.1 Blood for Exploratory Research ............................................................................. 59

11.6 Allelic Editing of CTX001 Drug Product ...................................................................... 59
11.7 Bone Marrow Aspirate ................................................................................................... 59
11.8 Sperm and Oocyte Banking ............................................................................................ 59
11.9 Patient Reported Outcomes ............................................................................................ 59
11.9.1 Pain-Scale (11-point NRS) ..................................................................................... 59
11.9.2 EQ-5D-5L/EQ-5D-Y .............................................................................................. 60
11.9.3 FACT-BMT ............................................................................................................ 60
11.9.4 ASCQ-Me ............................................................................................................... 61
11.9.5 PedsQL and PedsQL Sickle Cell Disease Module ................................................. 61
11.10 Mini-Mental State Examination ..................................................................................... 62
11.11 Wechsler Abbreviated Scale of Intelligence .................................................................. 62
11.12 Safety .............................................................................................................................. 62
11.12.1 Adverse Events ....................................................................................................... 62
11.12.2 Clinical Laboratory Assessments ........................................................................... 62
11.12.3 Physical Examinations and Vital Signs .................................................................. 64
11.12.4 Weight/Height ........................................................................................................ 64
11.12.5 Electrocardiograms ................................................................................................. 65
11.12.6 Contraception and Pregnancy ................................................................................. 65
11.12.6.1 Contraception ................................................................................................... 65
11.12.6.2 Pregnancy ......................................................................................................... 67
12 Statistical and Analytical Plans .......................................................................................... 68
12.1 Sample Size and Power .................................................................................................. 68
12.2 Analysis Sets .................................................................................................................. 68
12.3 Statistical Analysis ......................................................................................................... 68
12.3.1 General Considerations........................................................................................... 68
12.3.2 Background Characteristics .................................................................................... 69
12.3.2.1 Subject Disposition .......................................................................................... 69
12.3.2.2 Demographics and Baseline Characteristics .................................................... 69
12.3.2.3 Prior and Concomitant Medications................................................................. 70
12.3.3 Efficacy Analysis .................................................................................................... 70
12.3.3.1 Analysis of Primary Variables ......................................................................... 70
12.3.3.2 Analysis of Secondary Efficacy Endpoints ...................................................... 70
12.3.4 Safety Analysis ....................................................................................................... 71
12.3.5 Engraftment ............................................................................................................ 72
12.3.6 Adverse Events ....................................................................................................... 72
12.3.6.1 Clinical Laboratory Assessments ..................................................................... 73
12.3.6.2 Electrocardiogram ............................................................................................ 73
12.3.6.3 Vital Signs ........................................................................................................ 73
12.3.6.4 Physical Examination ....................................................................................... 73
12.3.7 Exploratory Analysis .............................................................................................. 73
12.3.8 Interim and Independent Data Monitoring Committee Analyses ........................... 74
12.3.8.1 Interim Analysis ............................................................................................... 74
12.3.8.2 Independent Data Monitoring Committee Analysis ........................................ 74
13 Procedural, Ethical, Regulatory, and Administrative Considerations ........................... 74

13.1 Adverse Event and Serious Adverse Event Documentation, Severity Grading, and
Reporting ........................................................................................................................ 74
13.1.1 Adverse Events ....................................................................................................... 74
13.1.1.1 Definition of an Adverse Event........................................................................ 74
13.1.1.2 Clinically Significant Assessments .................................................................. 75
13.1.1.3 Documentation of Adverse Events................................................................... 75
13.1.1.4 Adverse Event Severity .................................................................................... 76
13.1.1.5 Adverse Event Causality .................................................................................. 77
13.1.1.6 Study Drug Action Taken ................................................................................ 77
13.1.1.7 Adverse Event Outcome .................................................................................. 77
13.1.1.8 Treatment Given............................................................................................... 78
13.1.2 Serious Adverse Events .......................................................................................... 78
13.1.2.1 Definition of a Serious Adverse Event............................................................. 78
13.1.2.2 Reporting and Documentation of Serious Adverse Events .............................. 79
13.1.2.3 Expedited Reporting and Investigator Safety Letters ...................................... 80
13.2 Administrative Requirements ......................................................................................... 80
13.2.1 Ethical Considerations ............................................................................................ 80
13.2.2 Subject Information and Informed Consent ........................................................... 81
13.2.3 Investigator Compliance ......................................................................................... 81
13.2.4 Access to Records ................................................................................................... 81
13.2.5 Subject Privacy ....................................................................................................... 81
13.2.6 Record Retention .................................................................................................... 82
13.2.7 Study Termination .................................................................................................. 82
13.2.8 End of Study ........................................................................................................... 82
13.3 Data Quality Assurance .................................................................................................. 83
13.4 Monitoring ...................................................................................................................... 83
13.5 Electronic Data Capture ................................................................................................. 84
13.6 Publications and Clinical Study Report .......................................................................... 84
13.6.1 Publication of Study Results................................................................................... 84
13.6.2 Clinical Study Report ............................................................................................. 84
14 References ............................................................................................................................. 85
15 Protocol Signature Pages .................................................................................................... 89
15.1 Sponsor Signature Page .................................................................................................. 89
15.2 Investigator Signature Page ............................................................................................ 90
16 Appendices............................................................................................................................ 91
16.1 Busulfan PK Collection .................................................................................................. 91
List of Tables
Table 3-1 Study CTX001-121: Screening (Stage 1) ............................................................... 9
Table 3-2 Study CTX001-121: Pre-mobilization Period and Autologous CD34+ Stem Cell
Apheresis (Stage 1 and Stage 2) ........................................................................... 11
Table 3-3 Study CTX001-121: Myeloablative Conditioning and Infusion of CTX001
(Stages 3A/3B) ...................................................................................................... 14
Table 3-4 Study CTX001-121: Post-infusion Through Follow-up (Stage 4) ....................... 16
Table 3-5 Study CTX001-121: Transfusion Regimen .......................................................... 19
Table 9-1 Timing of the First Mobilization and Apheresis Cycle ........................................ 51

List of Abbreviations
Ab antibody
AE adverse event
Allo-HSCT allogeneic hematopoietic stem cell transplant
ALP alkaline phosphatase
ALT alanine transaminase
ANC absolute neutrophil count
ASCQ-Me Adult Sickle Cell Quality of Life Measurement System
AST aspartate transaminase
AUC area under the concentration versus time curve
bpm beats per minute
Cas9 CRISPR-associated 9 nuclease
CBC complete blood count
CI confidence interval
CRF case report form
CRISPR clustered regularly interspaced short palindromic repeats
crRNA crisprRNA
CSSCD Cooperative Study of Sickle Cell Disease
DLco lung diffusing capacity for carbon monoxide
DMSO dimethylsulfoxide
DMC data monitoring committee
DNA deoxyribonucleic acid
DSB double-strand break
EC ethics committee
ECG electrocardiogram
eCRF electronic case report form
EDC electronic data capture
EENT eyes, ears, nose, and throat
EQ-5D-5L EuroQol Quality of Life Scale – 5 dimensions – 5 levels of severity
EQ-5D-Y EuroQol Quality of Life Scale – 5 dimensions – youth
ETF early termination of follow-up
FACT-BMT functional assessment of cancer therapy-bone marrow transplant
FDA Food and Drug Administration
FSH follicle-stimulating hormone
G-CSF granulocyte colony-stimulating factor
GGT gamma-glutamyl transferase
GPS Global Patient Safety
GvHD graft-versus-host disease

GWAS genome-wide association studies
Hb hemoglobin
HBB human B globin
HBcAb hepatitis B core antibody
HbF fetal hemoglobin
HbS hemoglobin S
HbsAg hepatitis B surface antigen
HBV hepatitis B virus
HBVar Database of Human Hemoglobin Variants and Thalassemia Mutations
HCAb hepatitis C positive antibody
HCV hepatitis C virus
HDR homology-directed repair
hHSPCs human hematopoietic stem and progenitor cells
HLA human leukocyte antigen
HRQoL health-related quality of life
HSCT hematopoietic stem cell transplant
HU hydroxyurea
ICA internal carotid artery
ICF informed consent form
ICH International Council for Harmonization
INR international normalized ratio
IRB institutional review board
IV intravenous, intravenously
LDH lactate dehydrogenase
LT-HSC long-term hematopoietic stem cells
LVEF left ventricular ejection fraction
M month
max maximum value
MCA middle cerebral artery
MCH mean corpuscular hemoglobin
MCHC mean corpuscular hemoglobin concentration
MCV mean corpuscular volume
min minimum value

MRA magnetic resonance angiography
MRI magnetic resonance imaging
mRNA messenger RNA
MSH Multicenter Study of Hydroxyurea
N number of subjects
NAT nucleic acid testing
NHEJ non-homologous end-joining
NRS numerical rating scale
NSAIDs non-steroidal anti-inflammatory drugs
NSG NOD SCID gamma
nt nucleotide
PAM protospacer adjacent motif
PBMC peripheral blood mononuclear cells
PE physical examination
PedsQL Pediatric Quality of Life Inventory
PI principal investigator
PK pharmacokinetic(s)
PROs patient-reported outcomes
PT prothrombin time
PTT partial thromboplastin time
q6h every 6 hours
QC quality control
QRS the portion of an ECG comprising the Q, R, and S waves, together representing
ventricular depolarization
QT QT interval
QTcF QT interval corrected by Fridericia’s formula
RBC red blood cell
RDW red blood cell distribution width
RNA ribonucleic acid
RNP ribonucleoprotein complex
RPR rapid plasma reagin
SAE serious adverse event
SAP statistical analysis plan
SCD sickle cell disease
SD standard deviation

sgRNA single-guide RNA
SOP standard operating procedure
SUSAR suspected, unexpected, serious adverse reaction
TAMMV time-averaged mean of the maximum velocity
TCD transcranial Doppler
TDT transfusion dependent β thalassemia
TE treatment-emergent
tracrRNA trans-activating RNA
TRM transplant-related mortality
TRV tricuspid regurgitant jet velocity
ULN upper limit of normal
VAS Visual Analogue Scale
VOC vaso-occlusive crisis
WBC white blood cell
WHODD World Health Organization Drug Dictionary
Glossary of Terms
Term Definition
Engraftment Engraftment is defined as the first day of 3 measurements on 3 consecutive days with
absolute neutrophil count (ANC) ≥500/µL achieved within 42 days after CTX001
infusion and without use of unmodified CD34 + (backup) cells.
Enrollment Subject has signed consent AND is confirmed to be eligible
Intended genetic Intended genetic modifications are defined as indels that modify the sequence of the
modifications erythrocyte-specific enhancer in intron 2 of BCL11A
Severe SCD Severe SCD is defined by the occurrence of at least 2 severe VOCs per year during the
2-year period before screening, while receiving appropriate supportive care (e.g., pain
management plan, hydroxyurea)
Severe Severe VOC is defined as any 1 of the following events:
vaso-occlusive  Acute pain event that requires a visit to a medical facility and administration of pain
crises (VOCs) medications (opioids or IV NSAIDs) or RBC transfusions
 Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate
 Splenic sequestration, as defined by an enlarged spleen, left upper quadrant pain, and
an acute decrease in hemoglobin concentration of ≥2 g/dL.

5 INTRODUCTION
5.1 Background
5.1.1 Hemoglobinopathies
Hemoglobin (Hb) is a tetramer formed of 4 globin peptides, each tightly associated with a heme
group that contains an atom of iron. During gestation, the predominant form of hemoglobin is
fetal hemoglobin (HbF), which is composed of 2 α-globin chains and 2 γ-globin chains. Shortly
before birth, there is a switch from HbF to adult hemoglobin, which contains 2 α-globin and
2 β-globin polypeptide chains (Figure 5-1). The switch from HbF to adult hemoglobin is
mediated by a transcriptional switch from γ-globin to β-globin within the β-globin gene cluster
located on chromosome 11.
Figure 5-1 Polypeptide Chains in Adult Hemoglobin and Fetal Hemoglobin
Hemoglobinopathies are disorders caused by genetic defects that affect the production or
function of hemoglobin molecules. Two of the most common of the hemoglobinopathies are
sickle cell disease (SCD) and β-thalassemia.
5.1.2 Sickle Cell Disease
SCD is one of the most common monogenic disorders, affecting millions of people.1 It is
estimated to affect over 100,000 individuals in the US and about 42,000 individuals in Europe.2
The most severe and prevalent form of SCD, referred to as sickle cell anemia, is an autosomal
recessive disease due to homozygous mutations in which a valine replaces a glutamic acid at
position 6 in the β-globin protein, which leads to polymerization of deoxygenated hemoglobin
and red blood cell (RBC) sickling.
SCD is a chronic disease, characterized by recurrent acute vaso-occlusive crises (VOCs), which
lead to acute pain, chronic hemolysis, anemia, progressive tissue injury, and organ dysfunction.
The disease affects multiple organs causing acute and chronic complications such as acute chest
syndrome, stroke, priapism, splenic sequestration, osteonecrosis, renal failure, pulmonary
hypertension, liver disease, bone damage, limited growth, increased susceptibility to infections,
fatigue, and progressive cognitive decline.3, 4
About 90% of children born with SCD in the US or EU will survive into adulthood, but their
lifespan is shortened by 2 to 3 decades compared to the general population, with a median age of
death of approximately 40 to 50 years.5-9
Approved therapies to prevent complications of SCD include hydroxyurea (HU) in the US and
EU and L-glutamine oral powder in the US.10-12 These therapies reduce complications of SCD;

however, patients can still have breakthrough VOCs. Furthermore, HU is not effective in all
patients, is not well tolerated, and has carcinogenic and teratogenic risks. Allogeneic
hematopoietic stem cell transplant (HSCT) is the only known cure for SCD, but HSCT is only
available to about 20% of patients who have a matched donor13, and graft-versus-host disease
(GvHD) is a known risk.
Therefore, there is significant unmet medical need for the treatment of SCD.
Gene editing with CTX001 is intended to induce changes in the DNA sequence in the autologous
CD34+ hHSPC such that upon erythroid differentiation in the patient, the expression of γ-globin
is increased, leading to an increase in HbF expression levels in adult erythroid cells. The increase
in HbF is expected to ameliorate the clinical manifestations of SCD.
5.2 Therapeutic Rationale

The CRISPR-Cas9 editing therapeutic approach is to restore HbF production through editing of a
non-coding region in the BCL11A gene. BCL11A is a transcriptional silencer of γ-globin gene
expression and hence a negative modulator of HbF.
Several independent lines of evidence support the therapeutic hypothesis that an increase in HbF
will ameliorate the clinical manifestations of SCD:
 Analyses of the molecular interactions show that HbF can inhibit the abnormal
polymerization of hemoglobin S (HbS) that leads to RBC sickling (Figure 5-2).14, 15
Figure 5-2 Fetal Hemoglobin Protection of RBC Sickling

A) HbS Polymerization B) Prevention of HbS Polymerization by HbF
(A) HbS tetramers polymerize under deoxygenated conditions due to interactions between the mutated
valine at position 6 and a hydrophobic patch of an adjacent HbS tetramer at positions 85 to 88.
(B) The anti-sickling effect of HbF is due to the substitution of a less hydrophobic amino acid at position
87 (glutamine rather than threonine), which disrupts the hydrophobic pocket and prevents the interaction
of HbS tetramers to form polymers.
 Natural history and observational data support the protective role of HbF in the context of
naturally occurring variations:
o Infants with SCD are typically asymptomatic when their HbF levels are high, and only
become symptomatic when HbF synthesis is significantly reduced, typically at
approximately 6 to 9 months of age.14
o Populations that have polymorphisms associated with higher concentrations of HbF
(mostly in Saudi Arabia and Indian subcontinent) have milder forms of SCD.16

o Case reports and small studies show that people who are homozygous for the sickle
variant but also have high expression of HbF through adulthood (hereditary persistence of
fetal hemoglobin [HPFH]) have few or no SCD symptoms despite having HbS
concentrations >60%.17 In addition, people who are homozygous for deletional HPFH
type 1 or 2 and harbor large deletions including the β-globin genes are clinically
unaffected while they have 100% HbF.17,18
 HU, which is an approved pharmacologic therapy for the treatment of SCD, increases
production of HbF and ameliorates SCD symptoms.19
 Post hoc analyses of a randomized, placebo-controlled study of HU in SCD patients
(Multicenter Study of Hydroxyurea [MSH)] and an observational study of SCD patients
(Cooperative Study of Sickle Cell Disease [CSSCD]) show a strong association between
increased HbF and reduced pain rate.
5.2.1 CRISPR-Cas9 Gene Editing Approach
Gene editing using CRISPR-Cas9 can be used to create genetic modifications in CD34+ human
hematopoietic stem and progenitor cells (hHSPCs) with high specificity and frequency that will
result in a similar phenotype to the naturally occurring HPFH-associated variants, and are
expected to increase the production of HbF.
been repurposed as a RNA-guided DNA-targeting platform used for gene editing.20 It relies on
the DNA nuclease Cas9 and 2 noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA
(tracrRNA), to target the cleavage of DNA.
 crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through
Watson-Crick base pairing, typically with a 20-nucleotide (nt) sequence in the target DNA.
Changing the sequence of the 5' 20nt in the crRNA allows targeting of the CRISPR-Cas9
complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain
a sequence match to the first 20nt of the single-guide RNA (sgRNA) if the target sequence is
followed by a specific short DNA motif (with the sequence NGG) called a protospacer
 TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure that is
bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex
which can then cleave the target DNA.
 Once the CRISPR-Cas9 complex is bound to DNA at a target site, 2 independent nuclease
domains within the Cas9 enzyme will each cleave one of the DNA strands 3 bases upstream
of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA
terminate in a base pair (called a blunt end).
For the molecular reagents used in CTX001 production, the 2 RNA molecules (crRNA and
tracrRNA) are joined by a 4nt linker loop to form a chimeric RNA called sgRNA named SPY101
(Figure 5-3). The CRISPR-Cas9 (sgRNA/Cas9) complex together forms a ribonucleoprotein
complex (RNP) in situ.

Figure 5-3 Schematic of the CRISPR-Cas9 Complex
Complex containing a single gRNA wherein the crRNA and tracrRNA are joined by a linker loop (adapted from
Jinek20).
After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the
site-specific DSB, the next key step is repair of the DSB. Cells use 2 main DNA repair pathways
to repair the DSB: non-homologous end-joining (NHEJ) and homology-directed repair (HDR)
(Figure 5-4).
including non-dividing cells. NHEJ is error-prone and can often result in the removal or addition
of between 1 and several hundred nt at the site of the DSB, though such modifications are
typically <20nt.20,21 The resulting insertions and deletions (indels) can disrupt coding or
noncoding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA,
provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only
in dividing cells and occurs at a relatively low frequency in most cell types. NHEJ is the
preferable repair operant in our approach.

Figure 5-4 CRISPR-Cas9-Mediated Genome Editing Strategies
CRISPR-Cas9-mediated DNA cleavage creates a DSB at the target locus. The DSB can be repaired by NHEJ and
HDR (Genome Editing Strategy Image, 2015).
5.2.2 BCL11A Erythroid-lineage Specific Enhancer for Editing

BCL11A is a transcriptional silencer of γ-globin gene expression and hence a negative modulator
of HbF.22,23,24 Multiple lines of evidence from published studies support that targeting of
BCL11A will result in induction of HbF:
 In SCD transgenic mice, inactivation of BCL11A reverses γ-globin silencing and corrects
disease pathophysiology.25
 Genome-wide association studies (GWAS) identified variants in BCL11A that lead to an
increase in HbF and a reduction of the pain crisis rate in SCD patients.23
 Rare patients with microdeletions including the BCL11A locus (2p15-p16.1) show
persistence of HbF and normal hematologic and immunologic function.26
Screening of guide RNA (gRNA) molecules that lead to a phenocopy of HPFH effects led to the
identification of a guide (SPY101) that targets a critical erythroid-lineage specific transcription
factor-binding site (GATA1). The transcription factor-binding site is located within the
erythroid-lineage specific enhancer in the second intron of the BCL11A gene.
The erythroid specificity of this enhancer is supported by:
 In a transgenic mouse model carrying the human globin locus, BCL11A enhancer-deleted
mice (BCL11A Δenh) induce human γ-globin expression in erythroid cells, but do not exhibit
any other abnormalities including hematopoietic differentiation and cortical morphology.27 In
addition, while expression of BCL11A transcript was reduced >30-fold in erythroid cells of
BCL11A Δenh mice, there was no reduction of BCL11A expression in hHSPCs, B-cells or
cortical tissue.

 Engraftment studies in an immunocompromised mouse model using zinc finger nucleases to

disrupt the erythroid lineage-specific enhancer of BCL11A in human CD34+ cells showed
successful multilineage differentiation into lymphoid and myeloid cell lineages.28
 Data from 3 patients with microdeletions in a region that contains the BCL11A gene
(2p15-p16.1) show that in humans, normal hematopoiesis can occur despite a significant
reduction in the levels of BCL11A expression. These 3 patients, who had BCL11A
haploinsufficiency and a large reduction in BCL11A expression across mononuclear and
erythroid cell populations, also showed a large increase in HbF levels (16%, 24%, and 30%).
However, a variety of hematologic parameters were normal, including blood counts,
lymphocyte subtypes (assessed in 2 patients), and concentration of immunoglobulins
(measured in 1 patient).
 Engraftment data from nonclinical studies show that disruption of the BCL11A erythroid
lineage-specific enhancer using SPY101-RNP-edited human CD34+ cells did not impact
multilineage differentiation into lymphoid and myeloid cell lineages.
Because the repair process uses NHEJ, the gene editing will generate indels within the
non-coding BCL11A erythroid lineage-specific enhancer on chromosome 2, thus down-
regulating BCL11A in erythroid precursors with no effect expected in other hematopoietic
lineages. This noncoding change is expected to reactivate γ-globin gene transcription and elevate
HbF protein in RBCs (Figure 5-5).
Figure 5-5 Therapeutic Hypothesis for BCL11A Gene Editing

5.2.3 CTX001
CTX001 is a cellular product consisting of autologous CD34+ hHSPCs modified by
CRISPR-Cas9-mediated gene editing. The target of the CRISPR-Cas9 gene editing is the
erythroid lineage-specific enhancer region of the BCL11A gene located on intron 2 between
exons 2 and 3 on chromosome 2. The edits created with the highly specific guide, SPY101,
target a critical transcription factor binding site (GATA1) at the erythroid lineage-specific
enhancer region (identified as DNase I hypersensitive site +58, DHS+58) of the BCL11A gene.28-
30
The gRNA-Cas9 RNP (SPY101-RNP) introduces DSBs in DNA in a sequence-dependent
manner. Repair of the DSB by NHEJ results in DNA indels, intended to disrupt GATA1 binding,
thereby lowering BCL11A transcription, with concomitant increases in γ-globin and HbF levels.
Since SPY101-RNP precisely targets the non-coding erythroid lineage-specific enhancer region
of the BCL11A gene and not the BCL11A coding sequence itself, SPY101-RNP is expected
to modulate the levels of expression of the BCL11A gene and protein in cells solely of the
erythroid lineage and not affect non-erythroid hematopoietic lineages.
5.3 Nonclinical Experience
Details of the nonclinical efficacy and toxicology studies can be found in the Investigator’s
Brochure.
Briefly, in in vitro studies investigating CTX001, CRISPR-Cas9 gene editing at BCL11A gene
erythroid enhancer of healthy donor CD34+ cells led to a mean (SD) increase in γ/γβ-globin
mRNA ratios of 0.41 (0.15) and mean (SD) percentage of HbF/(HbF+HbA) protein levels of
29% (11%). Mean (SD) allele editing frequency was 80% (6%) and was uniform across
subpopulations of CD34+ cells, including long-term hematopoietic stem cells (LT-HSC). The
majority of editing was bi-allelic.
In mice xenotransplantation studies, there was no difference in engraftment between NOD SCID
gamma (NSG) mice infused with CTX001 and with control (mock or EGFP) edited CD34+
hHSPCs at 16 weeks post-transplantation. The mean (SD) frequency of edited alleles present in
the bone marrow samples at 16 weeks was 91% (15%). Furthermore, the engrafted cells were
able to maintain their multi-lineage potential.
In the nonclinical safety assessment of CTX001, on-target and potential off-target editing was
systematically evaluated using multiple orthogonal methods. CTX001 demonstrated a high rate
of on-target indels (approximately 88%) and no off-target editing compared with unedited
healthy donor cells. Karyotyping analysis did not identify any chromosomal translocations or
other detectable abnormalities.
A tumorigenicity study did not reveal any neoplastic or myeloproliferative lesions in the mice
receiving CTX001.
5.4 Clinical Experience
The current study is a Phase 1/2 study with CTX001 in subjects with SCD. CTX001 is also being
evaluated in subjects with transfusion dependent β-thalassemia (TDT) in Study CTX001-111. At
least 1 subject has been dosed with CTX001 in these studies and enrollment is ongoing.

6 STUDY OBJECTIVES
6.1 Primary Objective

 Evaluate the safety and efficacy of a single dose of autologous CRISPR-Cas9 modified
CD34+ hHSPCs (CTX001) in subjects with severe SCD
6.2 Secondary Objectives
 Assess the effects of infusion of CTX001 on disease-specific events and clinical status
 Quantify gene editing efficiency
6.3 Exploratory Objective
 Assess the ability of biomarkers to characterize CTX001 effect and predict treatment
outcomes
7 STUDY ENDPOINTS
7.1 Primary Endpoints

7.2 Primary Efficacy Endpoint
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for at least 3 months
7.2.1 Safety Endpoints
 Successful neutrophil engraftment within 42 days after CTX001 infusion
 Safety and tolerability assessments based on adverse events (AEs), clinical laboratory values,
and vital signs
 Transplant-related mortality (TRM) within 100 days after CTX001 infusion
 TRM within 1 year after CTX001 infusion
7.3 Key Secondary Efficacy Endpoint
 Relative change from baseline in annualized rate of severe VOCs starting 6 months after
CTX001 infusion
7.4 Secondary Endpoints
 Reduction in annualized rate of severe VOCs at the time of analysis from baseline by at least
50%, starting 6 months after CTX001 infusion
 Reduction in annualized rate of severe VOCs at the time of analysis from baseline by at least
65%, starting 6 months after CTX001 infusion

 Absence of severe VOCs for at least 12 months at the time of the analysis, starting 6 months
 Relative change from baseline in annualized duration of hospitalization for severe VOCs,
 Relative change from baseline in annualized rate of hospitalization for severe VOCs, starting
6 months after CTX001 infusion
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for at least 3 months,
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for at least 3 months,
starting at the time of CTX001 infusion, in the absence of treatment with HU
 Proportion of subjects with sustained HbF ≥20% at the time of analysis for 6 months, starting
6 months after CTX001 infusion, in the absence of treatment with HU
 Change in number of units of RBC transfused for SCD-related indications over time
 HbF concentrations over time
 Hb concentrations over time
leukocytes over time
 Proportion of alleles with intended genetic modification present in bone marrow cells over
time
 Change in patient reported outcomes (PROs) over time using;
o Pain-Scale:11-point numerical rating scale (NRS) – all subjects
o Functional assessment of cancer therapy-bone marrow transplant (FACT-BMT),
Adult Sickle Cell Quality of Life Measurement System (ASCQ-Me), and EuroQol
Quality of Life Scale (EQ-5D-5L) for ≥18 years
o EQ-5D-Youth (EQ-5D-Y; self-report and parent proxy), Pediatric Quality of Life
Inventory (PedsQL; Teen self-report and parent proxy), and PedsQL SCD Module
(Teen self-report and parent proxy): 12 to <18 years
7.5 Exploratory Endpoints
 Change in hemolytic index as measured by principal component analysis of 4 markers of
hemolysis (reticulocyte count, and serum concentrations of aspartate transaminase, lactate
dehydrogenase [LDH], and total bilirubin) over time
 Change in tricuspid regurgitant jet velocity (TRV) over time
 Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells) over
time
 Change in inflammatory and endothelial activation markers over time

8 STUDY POPULATION
Eligibility will be reviewed and documented by an appropriately qualified member of the

investigator’s team before subjects are enrolled.
Subjects who meet all of the inclusion criteria and none of the exclusion criteria will be eligible.
8.1 Inclusion Criteria
study:
1. Subject (or their legally authorized representative or guardian) will sign and date an informed
consent form (ICF) and, where applicable, an assent form.
2. Subjects 12 to 35 years of age, inclusive, on the date of informed consent.
3. Documented βS/βS or βS/β0 genotype. Subjects can be enrolled based on historical genotype
results, but confirmation of genotype is required before busulfan conditioning. The
β0 genotypes are defined using the HbVar Database.
4. Subjects with severe SCD. Severe SCD is defined by the occurrence of at least 2 of the
following events per year during the 2-year period before screening, while receiving
appropriate supportive care (e.g., pain management plan, HU):
 Acute pain events that requires a visit to a medical facility and administration of
pain medications (opioids or intravenous [IV] non-steroidal anti-inflammatory
drugs [NSAIDs]) or RBC transfusions
 Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate
 Splenic sequestration, as defined by an enlarged spleen, left upper quadrant pain,
and an acute decrease in hemoglobin concentration of ≥2 g/dL.
Historical severe VOCs will be adjudicated by the Endpoint Adjudication Committee (EAC).
5. Normal transcranial Doppler (TCD) velocity (time-averaged mean of the maximum velocity
[TAMMV] <170 cm/sec) in the middle cerebral artery (MCA) and the internal carotid artery
(ICA) for subjects 12 to 16 years of age.
6. Karnofsky performance status of ≥80% for subjects ≥16 years of age or Lansky performance
status of ≥80% for subjects <16 years of age.
9. Male subjects of reproductive capacity must agree to use effective contraception from start of
mobilization through at least 6 months after CTX001 infusion.

11. Willing to participate in the long-term follow-up study (Study VX18-CTX001-131), after
completion of this study.
8.2 Exclusion Criteria
Subjects meeting any of the following criteria are not eligible for enrollment:
2. Prior HSCT.
4. White blood cell (WBC) count <3 × 109/L or platelet count <50 × 109/L, not related to
hypersplenism per investigator judgment.
5. Treatment with regular RBC transfusions that, in the opinion of the investigator, cannot be
interrupted after engraftment.
6. Subjects with history of alloimmunization to RBC antigens and for whom the investigator
anticipates that there will be insufficient RBC units available for the duration of the study.
7. More than 10 unplanned hospitalizations or emergency department visits related to SCD in
the 1 year before screening that, in the opinion of the investigator, are consistent with
significant chronic pain rather than acute pain crises.
8. HbF level >15.0%, irrespective of concomitant treatment with HbF-inducing treatments such
as HU.
9. History of abnormal TCD (TAMMV ≥200 cm/sec) for subjects 12 to 18 years of age.
10. History of untreated Moyamoya disease or presence of Moyamoya disease at Screening that
in the opinion of the investigator puts the subjects at the risk of bleeding.
confound the results of the study or pose an additional risk to the subject. This may include,
but is not limited to: history of relevant drug allergies; history of cardiovascular or central
nervous system disease; history or presence of clinically significant pathology; history of
mental disease; or history of familial cancer syndrome.
14. Advanced liver disease, defined as
a. Alanine transaminase (ALT) >3 × the upper limit of normal (ULN) or direct bilirubin
value >2.5 × ULN or
b. Baseline prothrombin time (PT) (international normalized ratio [INR]) >1.5 × ULN, or
biopsy
15. Baseline estimated glomerular filtration rate <60 mL/min/1.73 m2

16. Lung diffusing capacity for carbon monoxide (DLco) <50% of predicted value (corrected for
hemoglobin and/or alveolar volume).
17. Left ventricular ejection fraction (LVEF) <45% by echocardiogram.
19. Intolerance, contraindication, or known sensitivity to plerixafor or busulfan. Prior
anaphylactic reaction with excipients of CTX001 product (dimethylsulfoxide [DMSO],
dextran).
20. Positive for the presence of human immunodeficiency virus-1 (HIV-1) or human
immunodeficiency virus-2 (HIV-2) (positive antigen/antibody AND nucleic acid tests
[NAT]), hepatitis B virus (HBV) (positive Hepatitis B core antibody [HBcAb] AND NAT
tests), syphilis (positive screening AND confirmatory tests), or hepatitis C virus (HCV;
positive antibody [HCAb] AND NAT tests). Additional infectious disease markers should be
obtained and tested as required by the local authority for the collection and processing of
cellular therapy products. These additional tests (e.g., HTLV-1, HTLV-2, malaria,
tuberculosis, toxoplasmosis, Trypanosoma cruzi, or West Nile virus) will be evaluated to
determine overall impact to the subject and manufacturing of CTX001.
21. Participation in another clinical study with an investigational drug/product within 30 days of
screening.
22. Subjects who are not able to comply with the study procedures outlined in the protocol as
judged by the investigator.
23. Pregnancy or breastfeeding.
9 STUDY IMPLEMENTATION
9.1 Study Design

This is a single-arm, open-label, multi-site, single-dose, Phase 1/2 study in subjects who have
severe SCD. The study will evaluate the safety and efficacy of a single dose of autologous
CRISPR-Cas9 modified hHSPCs (CTX001) and will initially include up to 17 subjects. The
subjects will be 12 to 35 years (inclusive) of age. The study may be expanded to include a total
of up to 45 subjects dosed.
As a safety measure during the initial stages of the study, the first 2 subjects will be treated with
before treating the second subject. The second subject will not undergo myeloablation until the
first subject achieves neutrophil engraftment (absolute neutrophil count [ANC] ≥500/µL for
3 consecutive days), the available engraftment and safety data have been reviewed by the data
monitoring committee (DMC), and at least 30 days after infusion of CTX001 to the first subject.
Once the second subject has achieved neutrophil engraftment and has not had Grade ≥3 AEs
other than those typically associated with busulfan conditioning or autologous transplant
procedure, the remaining subjects can undergo conditioning and CTX001 infusion concurrently.
If the second subject infused with CTX001 experiences Grade ≥3 AEs other than those typically
associated with busulfan conditioning or autologous transplant procedure through engraftment or

30 days post infusion, whichever is longer, a DMC meeting will be convened and data will be
reviewed before the remaining subjects can undergo conditioning and CTX001 infusion, and a
substantial amendment to the clinical trial application (CTA) will be submitted before the
remaining subjects can undergo conditioning and CTX001 infusion.
All steps that precede busulfan myeloablation such as consent, screening, and stem cell
collection may proceed concurrently without staggering subjects.
The study will initially enroll adult subjects, 18 to 35 years of age. The decision to enroll
adolescent subjects (12 to <18 years of age) will be based on a review of the engraftment, safety,
and efficacy data from the first adult subjects with of data following CTX001
infusion by the DMC. Approximately adult subjects will be dosed with CTX001 before the
conditioning and dosing of the first pediatric subject.
The decision to expand the study to include a total of up to 45 subjects dosed will be based on an
examination of the totality of the safety and efficacy data by the Sponsor in consultation with the
DMC and the Steering Committee (SC).
For each subject, the study will be conducted in 4 stages (Figure 9-1), which are described
below. All subjects who receive CTX001 will be asked to enroll into the long-term follow-up
study (Study VX18-CTX001-131).
Figure 9-1 CTX001-121 Study Design
9.1.1 Stage 1: Screening and Pre-mobilization Period

Screening Visit assessments are listed in Table 3-1 and assessment during the pre-mobilization
period are listed in Table 3-2.
The investigator (or an appropriate authorized designee at the study site) will obtain informed
consent from each subject. After informed consent, subject eligibility will be determined.
Fertility preservation
Subjects who meet the inclusion criteria will have the option to undergo fertility preservation via
cryopreservation of oocyte or sperm (this may occur at any time after eligibility is confirmed,
before initiation of busulfan conditioning). For pre-pubescent subjects, gonadal tissue collection
and cryopreservation may be performed per local practice and standard of care.
RBC transfusion
After eligibility is confirmed, subjects will begin RBC exchange transfusions for a minimum of
8 weeks before the planned start of mobilization and will continue receiving these transfusions

until they begin busulfan conditioning. The goal of these RBC transfusions is to maintain HbS
level of <30% of total Hb while keeping total Hb concentration ≤11 g/dL. Treatment with HU
should be stopped at least 8 weeks before planned mobilization.
9.1.1.1 Rescreening and Repetition of Screening Assessment(s)
Individuals who do not meet the eligibility criteria for participation upon initial screening (screen
failure) can be rescreened up to 2 times. All screening tests should be repeated to determine
eligibility except for: genotyping (if performed during the initial screening); DLco and
echocardiograph (if performed within 3 months), and infectious disease markers (if performed
within 60 days) unless judged by the investigator to be necessary. Brain MRI/MRA should be
repeated only if there are signs of neurological symptoms as judged by the investigator.
Repetition of individual screening assessment(s) that did not meet eligibility requirements is not
permitted with the following exceptions:
 If there is clear evidence of a laboratory error (e.g., hemolyzed sample) or equipment
malfunction, collection of a repeat sample for the appropriate laboratory test or assessment
may be permitted with the approval of the medical monitor.
 Individual laboratory results that are related to a temporary, reversible condition in the
opinion of the investigator may be retested once after the condition resolves or within
30 days, whichever is earlier.
If repeat values of the individual assessment(s) are within the eligibility criteria and completed
within the screening window, then the subject is eligible for the study.
screening process.
9.1.2 Stage 2: Mobilization, Autologous CD34+ Stem Cell Collection,
CTX001 Manufacture and Disposition
Assessments during mobilization and apheresis are listed in Table 3-2.
Before starting administration of plerixafor, subjects will be assessed by the study investigator to
confirm whether they are eligible to proceed with apheresis (as per local guidelines). If a subject
is deemed to not be eligible for mobilization and apheresis, this procedure can be delayed for up
to 3 months. Thereafter, the subject will be removed from the study.
Each subject will undergo stem cell mobilization with plerixafor only (see Section 9.8.2.1 for
details). Peripheral blood mononuclear cells (PBMC) will be collected by apheresis.
Subjects should receive an RBC exchange transfusion 3 (± 1) day before the start of
mobilization/ apheresis cycle.
On Day 1, subjects will receive plerixafor . Subjects will
undergo apheresis for up to 3 consecutive days to collect CD34+ hHSPCs (see Section 9.8.2.1 for
details). The targeted CD34+ cell collection is at least 15 × 106 CD34+ cells/kg for manufacturing
of CTX001 in order to achieve a minimum target dose of 3 × 106 CD34+ cells/kg. On Day 3 (if
required after Cycle 1), an additional 2 × 106 CD34+ cells/kg will be collected as backup for
rescue therapy in an event of non-engraftment with CTX001. Cell collection intended for
manufacturing will only occur on Days 1 and 2 of apheresis. On these days collected cells
intended for manufacturing will be shipped daily at 2°C to 8°C to the manufacturing facility.

9.1.3.1 Stage 3A: Myeloablative Conditioning

During CTX001 manufacturing and before the planned start of busulfan conditioning, subjects
will continue to receive RBC exchange transfusions with the goal of maintaining an HbS level of
<30% of total Hb while keeping total Hb concentration ≤11 g/dL for at least 8 weeks (Table 3-5).
If the planned start of busulfan conditioning is >4 months after completion of mobilization, the
investigator may stop the RBC transfusion regimen and restart HU for those subjects who have
been previously treated with HU. If the RBC transfusion regimen is interrupted, subjects should
begin RBC exchange transfusions a minimum of 8 weeks before the planned start of busulfan
conditioning with the goal of maintaining an HbS level of <30% of total Hb while keeping total
Hb concentration ≤11 g/dL.
Subjects should receive an RBC exchange transfusion 3 (± 1) days before the start of busulfan
conditioning (Table 3-5). After the CTX001 product is received at the site and it has been
confirmed that the backup cells remain available and in acceptable condition to be administered
if needed, the subject will begin busulfan conditioning.
Subjects will be assessed by the investigator to confirm their eligibility to proceed with
myeloablative conditioning before administration of busulfan. If a subject is deemed not to be
eligible for myeloablative conditioning, this procedure can be delayed for up to 3 months.
Thereafter, the subject will be removed from the study and the Sponsor will be notified.
The starting dose of busulfan will be 3.2 mg/kg administered IV once daily for 4 consecutive
days. However, busulfan may be administered as 0.8 mg/kg every 6 hours (q6h) for
4 consecutive days, per the site’s standard practice. Clinical sites may use a test dose of busulfan
30 (± 2) days before beginning myeloablation to pre-determine the busulfan dose. For subjects
less than 34 kg, institutional and/or regional practice may be used.
Busulfan pharmacokinetics (PK) will be collected on Day 1 and Day 3 of conditioning.
Additional details regarding busulfan PK are included in Appendix 16.1. The dose of busulfan
may be adjusted as required based on the PK of the first busulfan dose to maintain appropriate
levels for myeloablation. Additional details regarding busulfan administration are included in
Section 9.8.3.
 CTX001 integrity has been compromised in any way and is no longer suitable for
 The subject has any clinical condition that, in the opinion of the investigator, would put
If the busulfan infusion was given but the full dose was not administered, the investigator should
immediately inform the medical monitor.
9.1.3.2 Stage 3B: CTX001 Infusion (Day 1)
The CTX001 infusion will occur at least 48 hours after completion of the busulfan infusion and
no more than 7 days after completion of the busulfan infusion.
On Day 1, the entire dose of CTX001 (≥3 × 106 CD34+ cells/kg) will be infused through a
central venous catheter. Each vial containing CTX001 should be infused within 20 minutes after
thawing.

If the investigator suspects that CTX001 integrity has been compromised in any way and is no
investigator will not infuse CTX001 and will immediately contact the medical monitor.
9.1.4 Stage 4: Follow-up Through Engraftment and Up To 2 Years After
CTX001 Infusion
9.1.4.1 Stage 4A: Post-infusion In-hospital Follow-up (Engraftment)
Subjects will be monitored in the transplant unit and receive supportive care according to
standard practices for subjects undergoing HSCT. Subjects should receive RBC transfusions to
maintain Hb ≥7 g/dL (Table 3-5) and platelet transfusions to maintain platelets >50,000/L, or
otherwise follow institutional practices. Subjects will be monitored for AEs and engraftment.
Subjects will be discharged from the transplant unit upon neutrophil engraftment (ANC ≥500/μL
for 3 measurements on 3 consecutive days) and stabilization of major medical issues as per local
hospital guidelines and investigator judgment. Details on post-infusion infection prophylaxis and
surveillance are included in Section 9.8.5.
9.1.4.2 Stage 4B: Post-engraftment Follow-up
Stage 4B starts after subjects have successfully engrafted, are clinically stable, and have been
discharged from the transplant unit. Subjects will be followed for approximately 2 years after the
CTX001 infusion with physical examinations (PEs), laboratory and imaging assessments, and
AE evaluations.
Following engraftment, RBC transfusions should be avoided for subjects who have hemoglobin
levels ≥7 g/dL, unless medically indicated (e.g., symptomatic anemia or as a requirement for
surgery).
comprehensive PEs, and receive screening for malignancy based on appropriate country-specific
cancer guidelines and subject medical history. Subjects should also undergo appropriate
malignancy evaluation if they have unexplained symptoms, signs, or laboratory abnormalities
that could be related to an underlying malignancy. Examples include unexplained weight loss or
fever, lymphadenopathy, abnormal blood counts. The medical monitor should be notified if a
subject is diagnosed with a malignancy.
All subjects who receive CTX001 product will be asked to enroll in the long-term follow-up
study (Study VX18-CTX001-131), following completion or withdrawal/discontinuation from
Study CTX001-121. Data collected in the long-term follow-up Study VX18-CTX001-131, up to
2 years after CTX001 infusion, will be included in the analyses.
9.2 Data Monitoring Committee
A DMC will be formed before the first subject is dosed. The DMC’s objectives and operational
details will be defined in a separate document (DMC charter). The DMC will be charged with
ensuring the safety of the subjects, safeguarding their interests, and ensuring the quality and
integrity of the trial. The DMC will conduct reviews of study data as outlined in the DMC

charter. The DMC may recommend that the Sponsor suspend enrollment, amend the study, or
The DMC will review available engraftment and safety data for the first subject before the
second subject can undergo myeloablation. The DMC will also review data from the second
subject if the second subject infused with CTX001 experiences Grade ≥3 AEs other than those
typically associated with busulfan conditioning or the autologous transplant procedure through
engraftment or 30 days post infusion, whichever is longer, before the remaining subjects can
undergo myeloablation and CTX001 infusion.
The DMC will review the available engraftment, safety, and efficacy data from the first adult
subjects with of data following CTX001 infusion, before expanding enrollment
to subjects 12 to <18 years of age. Approximately adult subjects will be dosed with CTX001
before the conditioning and dosing of the first pediatric subject.
CTX001 in order to recommend whether or not to expand the study to include up to 45 subjects
dosed (expansion cohort).
The Sponsor or designee will be responsible for promptly alerting the DMC regarding any
suspected, unexpected, serious adverse reaction (SUSAR).
9.3 Steering Committee
9.4 Endpoint Adjudication Committee
The EAC will be composed of an independent, external group of experts with appropriate
clinical and scientific background to evaluate VOCs. The EAC will be adjudicating historical
VOCs (during the 2 years prior to enrollment) and on-study VOCs to ensure that the events meet
the study’s definition of a severe VOC. Historical VOCs that occur within the 2-year period,
including those which may begin just prior to the 2-year window and end during the 2-year
window, will contribute to the determination of eligibility. The EAC will adjudicate historical
VOCs to ensure that the number of historical VOCs are sufficient for eligibility. Details of the
EAC structure, function, and process will be included in the EAC charter.
9.5 Method of Assigning Subjects to Treatment Groups
This is an open-label study with a single treatment group.
9.6 Rationale for Study Elements
9.6.1 Study Design
The study is single-arm and open-label because of the need for stem cell transplant procedure to
deliver CTX001.
The overall process is consistent with procedures used for autologous HSCT in patients with
malignant diseases, with a few exceptions that are described below. Therefore, the risk
associated with the procedures in this study is not expected to be significantly different from the
standard risks of these procedures.

Prophylactic RBC exchange transfusions will be administered for a minimum of 8 weeks before
the planned start of mobilization through CTX001 infusion, to decrease the risk of VOCs during
mobilization and busulfan conditioning. The rationale for this transfusion guidance stems from
evidence demonstrating that transfusion of non-sickle RBCs by exchange techniques into
patients with SCD can mitigate physiologic complications such as stroke31, disease-related
complications after surgical procedures32, ACS33, and VOCs.34 Stem cell transplant protocols for
patients with SCD often involve RBC exchange transfusion to decrease HbS to <30% prior to
mobilization and conditioning.
Following mobilization with plerixafor alone, CD34+ stem cells will be collected by apheresis.
Collection of stem cells by apheresis rather than bone marrow harvest allows for easier isolation
of CD34+ cells as the process is less invasive for subjects and does not require general
anesthesia. The decision to use plerixafor alone instead of plerixafor in combination with
granulocyte colony-stimulating factor (G-CSF), which is the standard regimen used for
collection, comes from the fact that administration of G-CSF can induce severe VOCs in subjects
with SCD, which can be fatal.35 Data from studies in healthy subjects and subjects with
β-thalassemia show that plerixafor alone can effectively mobilize CD34+ cells.36,37, 38 These
studies also show that CD34+ cells collected using plerixafor alone in healthy subjects
successfully engraft when administered in the context of an allogeneic HSCT. Inability or
anticipated inability to successfully manufacture the target dose of CTX001 is among the
stopping criteria for this study.
Busulfan alone will be used for conditioning. For allogeneic stem cell transplantation, busulfan is
typically combined with cyclophosphamide or fludarabine, since this combination provides both
myeloablation and immunosuppression.39 For autologous transplantation, as in the proposed
clinical study with CTX001, immunosuppression to prevent GvHD is not necessary and single
agent busulfan will provide predominantly a myeloablative effect.40 Regimens using busulfan
conditioning have been used in allogeneic transplantation of SCD and have resulted in successful
engraftment, fewer treatment-related complications, and stable donor chimerism.41 In addition,
other gene therapy studies have successfully used conditioning with busulfan alone in disease
areas such as β-thalassemia, SCD, and severe combined immunodeficiency due to adenosine
deaminase deficiency.42, 43
The study will initially enroll only adult subjects, and enrollment will be expanded to adolescent
subjects after data are available from adults (see Section 9.1). There is a strong rationale for
including adolescents because of the similarities with adults in terms of etiology and
pathophysiology of SCD and expected response to treatment. Overall, the study procedures and
potential risks of HSCT with CTX001 are expected to be similar in adult and adolescent
populations.44-46
9.6.1.1 Primary Efficacy Endpoint
CTX001 is a cellular product developed specifically to increase the production of HbF in
erythrocytes. By measuring the levels of HbF in peripheral blood, the intended consequences of
administration of CTX001 will be directly assessed (Section 5.2).
The primary efficacy endpoint is the proportion of subjects with sustained HbF ≥20% at the time
of analysis for at least 3 months starting 6 months after CTX001 infusion, in the absence of
treatment with HU. A subject will be considered to have met the primary efficacy endpoint if the

complications of SCD; however, patients can still have breakthrough VOCs. HU is not effective
in all patients and its long-term benefit on organ dysfunction progression is not known.
ADAKVEO, recently approved in the US, is indicated for the reduction of VOC frequency in
adults and pediatric patients aged 16 years and older with sickle cell disease. Pivotal studies
showed a reduction in overall VOC events with treatment compared with placebo. OXBRYTA,
which is conditionally approved in the US, is indicated for the treatment of SCD in adults and
pediatric patients 12 years of age and older. Pivotal studies showed an overall improvement in
total hemoglobin levels in treated patients compared with placebo. Importantly, while these
therapies may show reductions in some disease manifestations, HLA-matched sibling allo-HSCT
is the only known cure for SCD but <20% of patients have a matched related donor and GvHD
remains a significant risk. Therefore, there is significant unmet medical need for the treatment of
patients with severe SCD.
This study will enroll subjects with severe SCD. Severe SCD is defined by the occurrence of at
least 2 of the following events each year during the 2-year period before screening, while
receiving appropriate supportive care (e.g., pain management plan, HU if indicated):
 Acute pain event that requires a visit to a medical facility and administration of pain
medications (opioids or IV NSAIDs) or RBC transfusions
 Acute chest syndrome, as indicated by the presence of a new pulmonary infiltrate associated
with pneumonia-like symptoms, pain, or fever
 Splenic sequestration, as defined by an enlarged spleen, left upper quadrant pain, and an
acute decrease in hemoglobin concentration of ≥2 g/dL.
Recurrent VOCs will serve as a clinical endpoint to allow assessment of the clinical benefits by
assessing the change in severe pain crises and other VOC manifestations.
Subjects with baseline HbF >15% will not be eligible for enrollment. Since very few patients
with SCD who have high HbF levels have recurrent severe VOCs, an upper limit for HbF was
included to support the fact that the events collected from the medical records are acute severe
VOCs related to SCD. It will also ensure that subjects meeting the primary efficacy endpoint will
have a meaningful increase in HbF levels.
Subjects who have a history of requiring regular RBC transfusions to prevent SCD complications
and who, in the judgment of the investigator, cannot stop RBC transfusions after CTX001
infusion will not be eligible for enrollment because in order to accurately assess the primary
efficacy of HbF ≥20% for at least 3 months, RBC transfusion will have to be stopped after
CTX001 infusion as specified per study protocol.
9.6.3 Dose
Autologous transplantation for various indications typically uses at least 2 to 2.5 × 106
CD34+ cells/kg to support engraftment.52-54 To ensure engraftment in all subjects in the SCD
study, the Sponsor selected a conservative minimum dose of 3 × 106 CD34+ cells/kg, which is
20% to 50% higher than the typical minimum dose for autologous transplantation. In principle,
infusion of a higher number of CD34+ stem cells after myeloablation is associated with more
rapid engraftment, durability, and efficacy of the treatment.55

Based on current manufacturing capabilities and projected cell yields, the Sponsor proposes to
set a maximum cell dose limit of 2 × 107 CD34+ cells/kg. The Sponsor, along with the DMC,
will monitor for any potential dose related toxicities.
9.6.4 Study Duration
The total duration of follow-up for subjects who receive CTX001 will be 15 years following
infusion.
Subjects will be followed on Study CTX001-121 for 2 years after CTX001 infusion. Because the
study will enroll subjects with at least 2 severe VOCs per year during the 2-year period before
screening, a 2-year follow-up will allow meaningful assessment of the decrease in the annualized
incidence of VOCs.
Additionally, all subjects who receive CTX001 will be asked to enroll in the long-term follow-up
study (Study VX18-CTX001-131), after completion or withdrawal/discontinuation from
Study CTX001-121. The planned duration of Study VX18-CTX001-131 is up to 15 years’
follow-up after CTX001 infusion. This is to ensure that any potential long-term AEs related to
CTX001 are captured and to evaluate long-term treatment outcomes as well as to provide a
longer follow-up for efficacy.
9.7 Prior and Concomitant Treatments and Procedures
9.7.1 Prior Medications
All medication taken within 30 days before signing of the ICF and assent (where applicable) will
be recorded.
Retrospective information on RBC transfusions will be recorded from 24 months prior to date of
consent and assent (where applicable).
9.7.2 Venous Access
infusion of CTX001. A central venous catheter may also be used for apheresis, for RBC
exchange transfusions, and for clinical care of the subject following CTX001 infusion as per
9.7.3 Prohibited Medications
HU: Treatment with HU should be discontinued at least 8 weeks before planned start of
mobilization. Subjects may be restarted on HU after mobilization and apheresis if deemed
necessary by the investigator and if >4 months are planned between completion of mobilization
and start of busulfan conditioning. If HU is restarted, treatment with HU should be discontinued
once RBC transfusions are restarted before busulfan conditioning.
After CTX001 infusion, once engraftment is achieved, HU may be restarted if the subject
experiences VOCs or other complications that are judged by the investigator to be related to
SCD and warrant re-initiation of HU. If HU is restarted, it is recommended that HU be
progressively tapered and discontinued, if considered medically acceptable.
L-Glutamine: Treatment with L-Glutamine should be discontinued after CTX001 infusion.
HbF-inducing agent (other than HU): Treatment with any HbF-inducing treatment should be
discontinued after CTX001 infusion.

G-CSF: G-CSF should not be administered after CTX001 infusion. G-CSF may be administered
if engraftment is not achieved by Day 21 after informing the medical monitor.
Iron Chelators: Chelation must be discontinued at least 7 days before the start of myeloablative
Iron chelation with deferasirox, deferoxamine, or deferiprone should not be started until at least
1 month after the CTX001 infusion to allow for stable hematopoietic recovery and avoid
potential myelosuppressive effects.
9.8 Administration
9.8.1 RBC Transfusion
Refer to Table 3-5 for details on RBC transfusion.
9.8.2 Mobilization and Apheresis
9.8.2.1 Mobilization
The decision on whether a central line is needed for mobilization will be made by the
apheresis-experienced nurse or physician. Mobilization will be with plerixafor only. G-CSF
should NOT be administered.
Subjects will receive plerixafor at a dose of 0.24 mg/kg via subcutaneous injection
Weight taken within 5 days before the first day of mobilization will
be used for calculating the recommended dose. If multiple weights have been taken during the 5
days prior to first day of mobilization, then the weight recorded closest to time of mobilization
should be used for calculation of the cell dose. Refer to Table 9-1 and Table 9-2 for the full
dosing schedule.
CD34+ cell count in peripheral blood will be performed as listed in Table 3-2.
Refer to the plerixafor package insert for product details. Refer to the apheresis manual for
further details on mobilization, apheresis, and infusion procedures.
Table 9-1 Timing of the First Mobilization and Apheresis Cycle

Plerixafor administration X X X
)a
CD34+ stem cell apheresis for CTX001 product Xb Xb Xc
and backup cells
a
Plerixafor will be administered only if apheresis collection is planned for that day.
b
Cells should be shipped to central manufacturer at the end of the first and second collection day.
c

Table 9-2 Timing of the Subsequent Mobilization and Apheresis Cycle (After the
First Mobilization/Apheresis Cycle)

Plerixafor administration X Xb Xb
a
CD34+ stem cell apheresis for CTX001 product Xc Xc Xd

and backup cells
a
Plerixafor will be administered only if apheresis collection is planned for that day.
b
On Days 2 and 3 of apheresis will be performed as needed.
c
Cells may be collected for manufacturing on Day 1 and Day 2 (as needed) if sufficient CD34 + cells are not
collected. CD34+ cells may be collected for backup on any day after sufficient CD34 + cells are collected for
manufacturing.
d
9.8.2.2 Apheresis Procedure

PBMCs will be collected per clinical site standard operating procedures (SOPs) for up to
3 consecutive days. The targeted CD34+ cell collection for manufacturing of CTX001 is at least
15 × 106 CD34+ cells/kg. An additional 2 × 106 CD34+ cells/kg will be collected as backup for
rescue therapy in an event of non-engraftment with CTX001. Target to process 22 liters
(approximately 4 total blood volumes) as allowed per local regulations. Collection of backup
cells must occur prior to proceeding to Stage 3 (busulfan conditioning).
CTX001 product and safety backup, or if a subject cannot complete apheresis due to VOC(s),
2 additional mobilization and apheresis cycles will be allowed to collect additional cells. The
additional mobilization cycle should be initiated at least 14 days after the first day of the prior
mobilization cycle. Based on the number of CD34+ cells received at the manufacturing site and
the manufactured CTX001 dose, the medical monitor will inform the clinical site if additional
mobilization cycle will be necessary. If the minimum cell dose target for manufacturing or
backup is not met after 3 mobilization cycles and subject has tolerated previous apheresis cycles,
an additional mobilization cycle may be allowed in consultation with the medical monitor.
Any AEs associated with the apheresis procedure should be managed as per the site’s standard
guidelines.
Cells should be shipped at the end of the first and the second collection day. Cells collected for a
backup dose will be cryopreserved and stored at the study site. Additional details and
instructions on shipment and receipt are included in the study reference manual.
9.8.3 Conditioning: Busulfan Administration
Busulfan will be administered IV daily at a starting dose of 3.2 mg/kg/day for 4 consecutive days
(based on weight collected within 3 to 7 days prior to the first day of busulfan administration).
Once-daily dosing is the preferred schedule, but the busulfan dose regimen may be adjusted to be
given q6h per site’s standard practice. A test dose of busulfan may be performed 30 (± 2) days

before beginning myeloablation to pre-determine the busulfan dose. For subjects less than 34 kg,
institutional and/or regional practice may be used (see Section 9.1.3.1).
Busulfan PK will be collected on Day 1 and Day 3 of conditioning. Additional details regarding
busulfan PK are included in Appendix 16.1. The dose of busulfan may be adjusted based on the
PK of the first busulfan dose to maintain appropriate levels for myeloablation. The average target
AUC for subjects at a starting dose of 3.2 mg/kg/day for 4 days is 5000 µMmin (range: 4500 to
5500), equivalent to a target cumulative busulfan exposure of 90 mghr/L (range 80 to
100 mg  hr/L). The AUC for subjects receiving busulfan q6h for 4 days is 1125 µMmin (range:
900 to 1350). Refer to Appendix 16.1 for the busulfan PK collection schedule.
9.8.4 CTX001 Infusion
CTX001 will be supplied in infusion vial(s). All vial(s) containing CTX001 should be infused.
Further description is provided in the reference manual.
9.8.5 Post-CTX001 Infusion Infection Prophylaxis and Surveillance
If a subject develops sepsis or systemic bacteremia following CTX001 infusion, appropriate
cultures and medical management will be initiated.
9.9 Enrollment Suspension Criteria and Stopping Rules
9.9.1 Enrollment Suspension Criteria
The investigator will immediately notify the medical monitor of any of the following:
 Fatal or life-threatening serious adverse events (SAEs)
 Malignancies
 Grade 4 VOCs associated with mobilization
 Failure to collect the target number of CD34+ cells
Any SAEs will also be reported to Vertex Global Patient Safety in accordance with
Section 13.1.2.2.
 Death (non-accidental) on study after CTX001 infusion.
 Detection of leukemia, myelodysplastic disease, or myeloproliferative disorder in a
subject who has received CTX001.

 Failure to achieve neutrophil engraftment after infusion of CTX001 in 2 subjects, as

cells.
 Any SAE at least possibly related to CTX001 and not related to busulfan in 3 subjects,
with the exception of
o Grade 3 elevation in ALT, aspartate transaminase (AST), alkaline phosphatase
(ALP), gamma-glutamyl transferase (GGT), or bilirubin that resolve to baseline or
within normal range in 10 days
 Occurrence of VOC associated with a Grade 4 treatment-related AE in 2 subjects, during
or immediately after stem cell mobilization and collection.
 Failure to manufacture the target cell dose of CTX001 in 3 of the first 6 subjects who
have undergone the maximum allowed mobilization and apheresis cycles.
Enrollment will be temporarily suspended for lack of efficacy if:

 HbF is <5% at 9 months in 3 of the first 12 subjects
 The proportion of alleles with intended genetic modification present in peripheral blood
leukocytes is <10% in 3 of the first 12 subjects at 2 consecutive time points after
will be reviewed. After evaluation, and following a review of the data with the DMC, the
Sponsor and the SC may decide to restart enrollment in the study, amend the study, or
permanently suspend enrollment and remove all subjects who have not received CTX001 from
the study. A substantial amendment will be submitted before resuming enrollment.
Sponsor and the SC may decide to restart enrollment, amend the study, or permanently suspend
will not undergo stem cell mobilization or myeloablation with busulfan, and will have an end of
followed up or transitioned to the long-term safety follow-up study (Study VX18-CTX001-131).
unedited CD34+ cells will be up to the investigator’s discretion after discussion of the potential

risks with the subject. The investigator should inform the medical monitor of the decision before
infusion of cellular product.
parties including principal investigators (PIs), Ethics Committees (EC), IRB, and competent
authorities.
9.9.2 Stopping Rules for Individual Subjects
 Any medical condition that, in the opinion of the investigator, would put the subject at
risk for continuing study-related procedures or follow-up
 If a subject becomes pregnant or begins breastfeeding before start of busulfan
conditioning
 Inability or anticipated inability to successfully manufacture the target cell dose of
CTX001.
 If a subject is found not to have met eligibility criteria or has an important protocol
deviation before the start of busulfan conditioning that, in the opinion of the investigator,
would put the subject at risk for continuing study-related procedures or follow-up.
 Failure to successfully manufacture CTX001
9.10 Removal of Subjects
A subject (or legally authorized representative) may withdraw consent to participate in the study
at any time. If a subject withdraws consent the date and reason for withdrawal should be
documented. Subject data collected up to the date of consent withdrawal will be included in the
analyses. If the subject withdraws consent for the study, no further evaluations will be
performed, and no additional samples will be collected. The Sponsor may retain and continue to
use any data and samples collected before such withdrawal of consent. Wherever possible, the
tests and evaluations listed for the end of study visit will be carried out. The Sponsor will be
notified of all study withdrawals.
If a subject withdraws consent to participate in the study after infusion of CTX001, the subject
will be asked to enroll in the long-term follow-up study (Study VX18-CTX001-131). If the
subject refuses to participate in the long-term follow-up study, the subject will be asked if they
consent (and assent, where applicable) to have their information collected through review of
medical records or quarterly telephone interviews (partial withdrawal of consent). All subjects
who withdraw after CTX001 infusion should be followed for safety whenever possible.
Other reasons for removal of a subject before start of busulfan conditioning include:
 Any medical condition that, in the opinion of the investigator, would put the subject at risk
for continuing study-related procedures or follow-up
 If a subject is found not to have met eligibility criteria or has an important protocol deviation
which compromises subject safety
 Subject becomes pregnant or begins breastfeeding

 Failure to successfully manufacture the minimum dose of CTX001 or deliver CTX001 to the
site
 Administrative decision by Sponsor
If a subject chooses to withdraw from the study after the start of busulfan conditioning and
before administration of CTX001, the subject’s stored, unedited backup stem cells will be
If a 10/10 HLA matched related donor becomes available after enrollment but before starting
busulfan conditioning, then the subject will be withdrawn from the study.
9.11 Replacement of Subjects
10 STUDY DRUG INFORMATION AND MANAGEMENT
10.1 Preparation and Dispensing

10.1.1 Manufacture of CTX001
cryopreservation in the vapor phase of liquid nitrogen (≤-135°C). QC testing and batch release
should normally be completed in . Product will then be shipped at ≤-135°C to the clinical
site in 1 or more 20-mL vials. All manufacturing procedures are carried out in GMP cleanrooms.
10.1.2 Shipment of CTX001 Product to Treatment Site
CTX001 (packaged in vial[s]) will be shipped cryopreserved to the clinical sites in sealed dry
nitrogen shippers (≤-135°C) validated for at least 10 days. On receipt, package integrity will be
10.1.3 Storage of CTX001 Product
CTX001 will be stored at the site in the frozen state at a temperature of ≤-135°C until just prior
manual.
10.1.4 CTX001 Infusion Procedures, Dose, and Administration
Dextran-40. Histamine release associated with DMSO can result in adverse effects including
nausea, vomiting, diarrhea, flushing, fevers, chills, headache, dyspnea, rashes, bronchospasm,
anaphylaxis, vasodilation and hypotension, and mental status changes. Given the risk of these
AEs, subjects will be pre-medicated with an antihistamine (diphenhydramine hydrochloride) and

an antipyretic (acetaminophen or paracetamol) before dosing with CTX001. These medications

may be repeated every 3 to 4 hours as needed as per institutional guidelines and investigator
judgment.
site SOPs. The entire dose of CTX001 should be infused within 20 minutes of thaw. Detailed
instructions for the thaw and infusion of cells are in the study reference manual. Hospital
guidelines will be used to maintain chain of custody for CTX001 from the stem cell laboratory to
the subject. Before infusion, local procedures should be followed regarding the verification of
subject identity and product details to ensure a match as well as integrity of the product.
In the unlikely event that CTX001 infusion does not occur within 7 days after the last dose of
busulfan, subjects should receive the backup CD34+ stem cells.
10.2 Drug Accountability
10.3 Disposal of Unused Drug
10.4 Blinding and Unblinding
11 ASSESSMENTS
The schedule of assessments is shown in Table 3-1, Table 3-2, Table 3-3, Table 3-4, and
Table 3-5.
In addition to study-related visits, subjects will be followed per institutional guidelines or as
deemed appropriate by the investigator.

11.1 Subject and Disease Characteristics

Demographics (e.g., sex, age, race, ethnicity), 2 years of transfusion history (simple or RBC
exchange transfusions), and 2 years of SCD-related complications and associated treatment
(including inpatient hospitalization and medical facility visits) will be recorded. SCD history
such as genotype and age of diagnosis will also be recorded. Past and present medical history,
including targeted medical history and additional medical history considered by the investigator
to be significant, will also be recorded.
11.2 Transfusions
Any transfusion received and reason for transfusion (mL and number of transfused units) will be
documented from the time of consent through the end of the study. Additionally, Hb
concentration before and after transfusion will be documented. HbS will be collected prior to and
after all transfusions, and on each day of mobilization/apheresis cycle.
11.3 Diffusing Capacity of the Lungs for Carbon Monoxide
DLco (corrected) will be assessed. Pulmonary function tests will be performed with the subject
in a seated position and should be performed per site’s SOPs for pre-transplant workup and using
a calibrated spirometer.
11.4 Imaging Assessments
A brain MRI/MRA will be performed (for assessment of Moyamoya disease) at screening. If a
brain MRI/MRA has been performed within 3 months before date of consent, those results may
be used, unless repetition of the assessment is clinically indicated.
TCD will be performed at Screening following the technique used in the Stroke Prevention Trial
in Sickle Cell Anemia study,56 in children 12 to 16 years old to determine eligibility.
A transthoracic cardiac echocardiograph (for assessment of LVEF, TRV, and E/e’) will be
performed and read by trained medical personnel. Subjects who develop signs/symptoms of
congestive heart failure at any point during the study will undergo LVEF measurement by
echocardiograph.
11.5 Blood for Biomarker Assessments
Blood samples will be collected for evaluation of biomarkers and their ability to characterize the
effect of CTX001 and predict treatment outcomes.
 Protein based biomarkers, including but not limited to (1) hemoglobin fractionation and
quantitation in peripheral blood to assess bulk HbF levels, (2) total hemoglobin
concentration, (3) the proportion of circulating erythrocytes expressing fetal hemoglobin
(F-cells), and (4) inflammatory (CRP, IL6, IL8) and endothelial activation markers
(E-Selectin, P-Selectin)1
leukocyte DNA.

11.5.1 Blood for Exploratory Research

Blood samples will be collected for potential exploratory research to better understand CTX001
and its impact on hemoglobinopathies.
Samples may be used for exploratory research to identify molecular (genomic, metabolic and/or
proteomic) biomarkers that may be indicative of clinical response, resistance, safety,
pharmacodynamic activity (e.g., anti-Cas9 antibodies in cases of significant [>50%] HbF
decrease), and/or the mechanism of action of treatment. Samples of apheresed stem cells and
CTX001 will be collected during manufacturing for potential future genome analysis in case of
development of malignancy thought to be related to CTX001 and for exploratory research to
better understand CTX001 and its impact on SCD. Samples will not impact a subject’s minimum
final CTX001 dose.
11.6 Allelic Editing of CTX001 Drug Product
The proportion of alleles with intended genetic modification present in each batch of CTX001
will be assessed.
11.7 Bone Marrow Aspirate
Bone marrow aspirate samples will be collected for allelic editing testing to assess the proportion
of alleles with the intended genetic modification present in bone marrow DNA.
Samples may also be used for potential future genome analysis in case of development of
malignancy thought to be related to CTX001. Additionally, samples may be used for exploratory
research to identify molecular (genomic, metabolic and/or proteomic) biomarkers that may be
indicative of clinical response, resistance, safety, pharmacodynamic activity, and/or the
mechanism of action of treatment.
Detailed procedures for processing and handling bone marrow aspirate samples will be provided
in a separate document.
11.8 Sperm and Oocyte Banking
choose to undergo the procedures (or if less than 18 years of age, their legally authorized
representative who chooses to have them undergo the procedures) will do so after eligibility is
confirmed and prior to busulfan conditioning (Stage 3A). For pre-pubescent subjects, gonadal
tissue collection and cryopreservation may be performed per local practice and standard of care.
11.9 Patient Reported Outcomes
PRO assessments should be performed as the first assessment after obtaining informed consent
(and assent, where applicable), as well as be completed by subjects at the beginning of a study
visit prior to any assessments.
11.9.1 Pain-Scale (11-point NRS)
Numerical rating scale is a 1-dimensional measure of reporting intensity of pain in adults and
adolescents. The 11-point NRS is a segmented visual analogue scale (VAS) including numbers

from 0 to 10, ‘0’ representing no pain to ‘10’ representing worst possible pain. Each respondent
will select a whole number on the scale that reflects their pain intensity.
11.9.2 EQ-5D-5L/EQ-5D-Y
The EuroQol Questionnaire – 5 dimensions – 5 levels of severity (EQ-5D-5L) assesses an adult
subject’s health status in a standardized way and consists of 2 parts: the EQ-5D descriptive
system and the EQ VAS.
The EQ-5D-5L descriptive system comprises the same 5 dimensions: mobility, self-care, usual
activities, pain/discomfort, and anxiety/depression. Each dimension has 5 levels: no problems,
The EQ VAS records the subject’s self-rated health on a 100-point VAS with endpoints labeled
For subjects under the age of 18, child-friendly versions of EQ-5D-L are available for self
(EQ-5D-Youth version; EQ-5D-Y) and parent proxy completion (EQ-5D-Y proxy version). In
this study, the EQ-5D-Y will be administered for self-completion to subjects 12 to <18 years of
age and the parent proxy version for 12 to <18 years of age subjects who are unable to fill in the
questionnaire themselves. Subjects who are <18 at Screening should continue to complete the
EQ-5D-Y or parent proxy version for the entire study; they will not change to the adult version
(EQ-5D-5L) when they reach 18 years of age.
The EQ-5D-Y consists of 2 pages, the EQ-5D-Y descriptive system and the EQ VAS. The
descriptive system comprises the same 5 dimensions as the EQ-5D-5L but using a child-friendly
wording. Unlike EQ-5D-5L which utilizes 5 levels, each dimension in the Youth version has
3 levels: no problems, some problems, a lot of problems. The respondent is asked to indicate
his/her health state by ticking (or placing a cross) in the box against the most appropriate
statement in each of the 5 dimensions. The EQ VAS used in the Youth version is identical to the
EQ VAS used in EQ-5D-5L. Similar to EQ-5D-5L, responses can be used to determine a
quantitative measure of health outcome (i.e., health utilities).
11.9.3 FACT-BMT
The Functional Assessment of Cancer Therapy – for adult subjects undergoing bone marrow
physical, social, family, emotional, and functional well-being. The FACT-BMT consists of the
FACT-General (constitutes the core of all subscales) and treatment-specific concerns of bone
marrow transplantation.
Each statement in the FACT-BMT has a 5-point Likert-type response scale ranging from 0 to 4
(0 = “not at all”; 1 = “a little bit”; 2 = “somewhat”; 3 = “quite a bit”; and 4 = “very much”). The
subject is asked to circle or mark 1 number per line to indicate his/her response to the statement
as it applies to the past 7 days. Questionnaires are then scored, and the higher the score, the
better the QOL.

11.9.4 ASCQ-Me
ASCQ-Me is a disease-specific health-related quality of life (HRQoL) questionnaire that
measures physical, mental and social health along with information on severity of disease in
adult SCD patients. It includes the following domains: emotional impact, pain impact, pain
episodes, sleep impact, social functioning impact, stiffness impact, and SCD medical history
checklist. Most dimensions have 5 levels: never, rarely, sometimes, often, and always or not at
all, little bit, somewhat, quite a bit, and very much. Questions on the SCD medical history
checklist are indicated by yes or no options, and pain episode frequency and severity are
indicated by frequency of events. ASCQ-Me domains are scored using a T-score metric with a
mean of 50 for reference population and an SD of 10. Scores can be interpreted by considering
the direction of scoring and the difference between the reported score and the mean of 50 in
reference population (SD 10).
11.9.5 PedsQL and PedsQL Sickle Cell Disease Module
As a pediatric version of FACT-BMT does not exist, the Pediatric Quality of Life Inventory
questionnaire (PedsQL - Teen version) will be administered to subjects 12 to <18 years of age
for self-completion. A parent proxy version is available for subjects who are unable to complete
the questionnaire themselves. Subjects who are <18 at Screening should continue to complete the
PedsQL or parent proxy version for the entire study and will not complete the FACT-BMT when
they reach 18 years of age.
The PedsQL is a brief, standardized, generic instrument that systematically assesses subjects' and
parents' perceptions of HRQoL in pediatric subjects with chronic health conditions using
pediatric cancer as an exemplary model.57 The teen version of PedsQL (self-report and parent
proxy versions) comprises of 23 items across 4 domains including Physical Functioning,
Emotional Functioning, Social Functioning, and School Functioning.58
The PedsQL Sickle Cell Disease Module (PedsQL SCD; Teen version) is a disease-specific
module of PedsQL that will be administered to subjects 12 to <18 years of age for self-
completion in place of ASCQ-Me (validated only in adults). Subjects should continue to
complete the PedsQL SCD module or parent proxy version for the entire study and will not
complete the ASCQ-ME when they reach 18 years of age. The tool measures self-reported
HRQoL in SCD subjects across 9 domains (43 items in total): Pain and Hurt, Pain Impact, Pain
Management and Control, Worry I, Worry II, Emotions, Treatment, Communication I, and
Communication II. A parent proxy version is also available for subjects who are unable to
complete the questionnaire themselves.59
When completing the PedsQL and PedsQL SCD Module questionnaires, respondents are asked
to report the degree to which each item has been a problem over the past month by selecting the
most appropriate response on a 5-point scale ranging from 0 (never a problem) to 4 (almost
always a problem). Responses are transformed to a 0 to 100 score, with higher scores reflecting
better HRQoL.

11.10 Mini-Mental State Examination

Adult subjects will be evaluated for cognitive function using the Mini-Mental State Examination
(Version 2) after eligibility is confirmed and before start of first RBC exchange transfusion.
11.11 Wechsler Abbreviated Scale of Intelligence
Subjects (12 to <18 years of age) will be evaluated for cognitive ability using the 2-subtest form
of the second edition Wechsler Abbreviated Scale of Intelligence after eligibility is confirmed
and before the start of first RBC exchange transfusion.
11.12 Safety
Safety evaluations will include engraftment, TRM, AEs, clinical laboratory assessments, clinical
evaluation of vital signs, ECGs, and PEs.
Section 13.1 outlines the definitions, collection periods, criteria, and procedures for
11.12.2 Clinical Laboratory Assessments
Blood and urine samples will be collected according to Table 3-1 through Table 3-4. Additional
clinical laboratory tests may be obtained at any time as clinically necessary for safety purposes at
the investigator’s discretion.
Females of childbearing potential (as defined in Section 11.12.6) must have a negative
pregnancy test result at screening, within 3 days prior to starting mobilization, and within 5 days
before the initiation of busulfan conditioning.
The laboratory test panels are shown in Table 11-1.


Hematology Serum Chemistry Urinalysis Other Testsa
CBC with differential including: ALT Protein At screening only: Genotyping

Hemoglobin AST Blood of HBB and alpha loci (central
WBC Bilirubin (total and Nitrite laboratory)
ANC direct) Leukocyte
Platelet count Albumin esterase Hemoglobin fractionation
Reticulocyte count Alkaline phosphatase (central laboratory)
Nucleated RBCs Bicarbonate
MCV (Mean corpuscular BUN Allelic editing (blood; central
volume) Calcium laboratory)
MCHC (Mean corpuscular Chloride
hemoglobin concentration) Creatinine Allelic editing (bone marrow
MCH (Mean corpuscular Glucose aspirate; central laboratory)
hemoglobin) Magnesium
RDW (Red blood cell Phosphorous HbF distribution, F-cells
distribution width)a Potassium (central laboratory)
Sodium
Total protein CD34+ cell count
HbS
Inflammatory and endothelial

activation markers (central
laboratory)
If applicable:
Serum pregnancy test


Infectious Disease Marker Immunological Testing Coagulation Hemolysis Marker
Testingb
HBV (HbcAb, HbsAg, and CD4 PT Haptoglobin
NAT) CD8 (Prothrombin Lactate dehydrogenase
CD19 time)
HCV (HCAb and NAT)
CD16 aPTT
HIV-1, HIV-2 c CD56 (Activated
(Antigen/Antibody IgG partial
immunoassay, if positive, then IgD thromboplastin
NAT) IgE time)
IgM INR
Syphilisd IgA
HTLV-1 Abe
a
While a subject is on a regular transfusion regimen, samples for central labs should be collected immediately
before transfusion, and must be collected within 7 days before the next scheduled transfusion. After dosing with
CTX001, if the subject is no longer receiving regular transfusions, samples should be collected immediately
before a transfusion and at least 2 weeks after a previous transfusion.
b
Additional infectious disease markers testing should be done if required by local practice (e.g., HTLV-1, HTLV-
2, malaria, tuberculosis, toxoplasmosis, Trypanosoma cruzi, or West Nile virus).
c
In line with standard practice for donor screening, reflexive HIV testing is permitted.
d
Subjects must have at least a screening assay for syphilis performed (e.g., rapid plasma reagin [RPR] is
preferred). If positive, a confirmatory test (e.g., FTA-ABS, TPPA) must be done to confirm the presence of active
infection. Local testing algorithms for syphilis may be used, provided they contain these tests.
e
Testing for HTLV-1 Ab may be required depending on subject’s history and characteristics.
11.12.3 Physical Examinations and Vital Signs

A PE includes a review of the following systems: head, neck, and thyroid; eyes, ears, nose, and
throat (EENT); respiratory; cardiovascular; lymph nodes; abdomen (including spleen); skin;
musculoskeletal; neurological systems, and performance status. Breast, anorectal, and genital
examinations will be performed when medically indicated. Performance status will be assessed
using the Karnofsky Performance Scale for subjects ≥16 years old and the Lansky Performance
Scale for subjects <16 years old. After screening, any clinically significant abnormal findings in
PEs will be reported as AEs.
The abbreviated PE will include an assessment of the following body systems: head, neck, and
thyroid; EENT; cardiovascular system; respiratory system; skin; abdomen (including spleen),
lymph nodes; and neurological systems.
Vital signs include blood pressure (systolic and diastolic), temperature, pulse rate, pulse
oximetry, and respiration rate. The subject will be instructed to rest for at least 5 minutes before
vital signs are assessed.
11.12.4 Weight/Height
Subject weight (kg) and height (cm) will be measured with shoes off.

11.12.5 Electrocardiograms
Standard 12-lead ECGs will be performed using a machine with printout. Additional standard
12-lead ECGs will be performed at any other time if clinically indicated. The performance of all
ECGs will adhere to the following guidelines:
 The ECG will be done before any other procedures that may affect heart rate, such as blood
draws.
 The subject will be instructed to rest for at least 5 minutes before having an ECG.
 The test should be performed in the supine position.
A printout of the ECG traces will be made for safety review by the investigator or qualified
designee and maintained with source documentation. Clinically significant ECG abnormalities
occurring during the study through the Safety Follow-up Visit will be recorded as AEs.
11.12.6 Contraception and Pregnancy
11.12.6.1 Contraception
infusion.
Non-sterile male subjects of reproductive capacity who are or may become sexually active with
female partners of childbearing potential must agree to use acceptable, highly effective methods
of contraception to avoid fathering a child from start of mobilization through at least 6 months
Acceptable and highly effective methods of contraception for subjects and their partners are
listed below. If applicable, additional contraception requirements may need to be followed
according to local regulations and/or requirements.
 True abstinence from heterosexual intercourse for the subject, when this is in line with the
preferred and usual lifestyle of the subject. Periodic abstinence (e.g., calendar, ovulation,
symptothermal, post-ovulation methods) and withdrawal are not acceptable methods of
contraception.
 If the male is infertile (e.g., after bilateral orchiectomy) or pre-pubescent. Infertility may be
documented through examination of a semen specimen before mobilization.
 If the female is of non-childbearing potential, as described below.

Female Subjects
 Female bilateral tubal ligation performed at least 6 months previously.
 Female continuous use of an intrauterine device (non-hormone releasing or hormone
 Female combined (estrogen and progestogen-containing) or progestogen-only oral hormonal
contraception associated with inhibition of ovulation if successfully used for at least 60 days
before mobilization or with a second form of approved contraception for at least 60 days
after beginning hormonal contraception.
 Female subjects undergoing fertility preservation as described in Section 9.1.1 and who are
unable to use hormonal contraception during this time must use true abstinence during the
period of fertility preservation unless meeting another criterion listed as waivers of
contraception for the couple below.
Acceptable contraceptive methods for male partners of female subjects of childbearing potential:
 Condom with spermicide (either as a single product if commercially available and/or allowed
according to local regulations; otherwise condom and spermicide as separate products). Local
regulations may require use of an additional acceptable method of contraception.
 Vasectomy (with a negative sperm post-vasectomy semen analysis) at least 6 months before
start of mobilization, and 1 barrier method of contraception.
Male Subjects
 Condom with spermicide (either as a single product if commercially available and/or
contraception.
 Vasectomy (with a negative sperm post-vasectomy semen analysis) at least 6 months
 Bilateral tubal ligation performed at least 6 months previously.
 Continuous use of an intrauterine device for at least 90 days before start of mobilization.
 Hormonal contraceptives, if successfully used for at least 60 days before start of
mobilization.
Additional notes:
 Female condom cannot be used with male condom (as a double method of contraception)

 The use of birth control methods does not apply if the female partner has had a bilateral
 Male subjects who are not sexually active at the time of screening must agree to follow
 If over the course of the study the subject’s status changes and the subject does not meet
the criteria for waiving the contraception requirements, the subject must begin following
the contraceptive methods listed above.
 If applicable, additional contraception requirements may need to be followed according
 Male subjects must not donate sperm after start of mobilization, throughout the study,
 Unique situations that may not fall within the above specifications may be discussed with
Female Subjects of Non-childbearing Potential:
criteria:
 Postmenopausal: Female subjects who have been amenorrheic for at least 2 years and have a
serum follicle-stimulating hormone (FSH) level within the laboratory's reference range for
postmenopausal females
 Documented hysterectomy and/or bilateral oophorectomy
 Subject is premenarchal.
Note: All other female subjects (including subjects with tubal ligations and subjects who do not
have a documented hysterectomy) will be considered to be of childbearing potential.
11.12.6.2 Pregnancy
Subjects will be counseled to inform the investigator of any pregnancy that occurs during the
study. If a subject becomes pregnant during the study, study interventions that may put the fetus
at risk will be stopped immediately.
reported to the medical monitor and Vertex Global Patient Safety (GPS) within 24 hours of the
site’s knowledge using the Pregnancy Information Collection Form.
obtained. A separate ICF will be provided to explain these follow-up activities. Birth outcomes

enrollment. Baseline will be calculated using records of all severe VOCs adjudicated by the EAC
as meeting the protocol definition of severe VOC, during the 2 years prior to enrollment.
All severe VOCs that occur at least 6 months after CTX001 infusion until the end of study
(Month 24) will be captured as post-CTX001 severe VOCs. Only severe VOCs adjudicated by
the EAC as related to the underlying SCD and not to an acute intercurrent event, such as acute
bleeding or infection, will be included for evaluating the change from baseline in annualized rate
of VOCs.
Change (absolute change) from baseline will be calculated as Post-baseline value − Baseline
value.
Treatment-emergent (TE) Period will include the time from CTX001 infusion to last study
visit.
evaluated as observed. For subjects who are lost to follow-up or die, safety and efficacy analyses
will be based on their available data, before death or loss to follow-up. For subjects who
withdraw from this study but enroll in the long-term follow-up study (Study
VX18-CTX001-131), efficacy and safety will be based on data from both the parent and the
long-term follow-up studies up to 2 years after CTX001 infusion, unless otherwise specified.
Subgroup analyses, such as descriptive summaries of the key efficacy / safety endpoints by age
groups as well as by genotypes, will be provided as appropriate. Details will be described in the
SAP.
12.3.2 Background Characteristics
12.3.2.1 Subject Disposition
The number and percentage of subjects in the following categories will be summarized as
appropriate based on the Enrolled Set:
 Subjects who are mobilized
 Subjects who are dosed with CTX001
 Subjects who are dosed with CTX001 with at least 1 year of follow-up post-CTX001
infusion
 Completed study (24-month post-CTX001 follow-up)
 Prematurely discontinued the study and the reasons for discontinuation
12.3.2.2 Demographics and Baseline Characteristics
 Age, sex, race, ethnicity, weight, height, genotype
 Targeted medical history

 Results of screening imaging (cardiac) and selected laboratory tests

 Number and annualized rate of severe VOCs for past 2 years
12.3.2.3 Prior and Concomitant Medications
Medications used will be coded using the World Health Organization Drug Dictionary
(WHODD):
Prior medication: any medication that started before CTX001 infusion.
Concomitant medication: medication continued or newly received on or after CTX001
infusion.
A given medication may be classified as prior, concomitant, or both prior and concomitant.
Prior medications and concomitant medications will be summarized descriptively using
frequency tables by Preferred Name.
12.3.3 Efficacy Analysis
12.3.3.1 Analysis of Primary Variables
Details of the analyses will be provided in the SAP.
Analysis of Primary Efficacy Endpoint
The response status of the primary efficacy endpoint for a subject is evaluated with HbF values
obtained from the central laboratory measured at least 6 months after CTX001 infusion and until
the time of analysis. Response status for subjects who are lost to follow-up or die will be based
on their available HbF data, before death or loss to follow-up. Response status for subjects who
withdraw from the study but enroll in the long-term follow-up study
(Study VX18-CTX001-131), will be based on their HbF data from both the parent and the long-
term follow-up studies up to 2 years after CTX001 infusion. For subjects who receive HU, the
HU treatment period will be defined as the period from the first HU dose to 3 months after the
last HU dose. HbF data collected during the HU treatment period will be excluded in the analysis
of the primary efficacy endpoint.
A subject will be considered to have met the primary efficacy endpoint if, at the time of analysis,
HbF level is ≥20%, and if this HbF level ≥20% has been sustained for a period of at least
3 months in the absence of HU.
The proportion of subjects who meet the primary efficacy endpoint will be provided, with the
1-sided P value and the 2-sided exact Clopper-Pearson CI.
12.3.3.2 Analysis of Secondary Efficacy Endpoints
VOCs occurring from the time of screening to the first 6 months after CTX001 infusion will not
be included in any of the analyses of VOCs.
Key Secondary Endpoint
Relative change from baseline in annualized rate of severe VOCs will be calculated for each
subject and will be summarized.

Secondary Efficacy Endpoints

 Proportion of subjects with reduction in annualized rate of severe VOCs at the time of
analysis from baseline by at least 50% after CTX001 infusion with the corresponding 2-sided
95% exact Clopper-Pearson CI will be provided.
 Proportion of subjects with reduction in annualized rate of severe VOCs at the time of
analysis from baseline by at least 65% after CTX001 infusion with the corresponding 2-sided
95% exact Clopper-Pearson CI will be provided.
 Proportion of subjects with absence of severe VOCs for at least 12 months at the time of
analysis, with the corresponding 2-sided 95% exact Clopper-Pearson CI will be provided.
 Relative change from baseline in annualized duration of hospitalization for severe VOCs will
be summarized.
 Relative change from baseline in annualized rate of hospitalization for severe VOCs will be
summarized.
 Proportion of subjects achieving HbF ≥20% at the time of analysis for at least 3 months,
starting 3 months after CTX001 infusion at the time of analysis, with the 2-sided 95% exact
Clopper-Pearson CI will be provided.
starting at the time of CTX001 infusion at the time of analysis, with the 2-sided 95% exact
Clopper-Pearson CI will be provided.
starting 6 months after CTX001 infusion, with the 2-sided 95% exact Clopper-Pearson CI
will be provided.
 HbF and Hb concentrations will be summarized as a continuous variable over time.
 Relative change in number of units of RBC transfused for SCD-related indications will be
summarized.
 Change in PROs will be summarized as a continuous variable over time.
 Proportion of alleles with intended genetic modification present in bone marrow cells will be
12.3.4 Safety Analysis
Analysis based on the Safety Analysis Set will include:
 Proportion of subjects with neutrophil engraftment within 42 days after CTX001 infusion
 AEs, laboratory values, and vital signs from signing of informed consent through the
Month 24 visit.
 Incidence of TRM within 100 days and 1 year post CTX001 infusion. TRM defined as death

The number and percentage of subjects with AEs and SAEs will be summarized by severity and
seriousness in a tabular fashion according to the time periods defined in Table 13-1. The
percentage of subjects with AEs and SAEs within each time period will be based on the subset of
the Safety Set being followed beyond the start of that time period.
Time to neutrophil engraftment, defined as the first of 3 measurements on 3 consecutive days
with ANC ≥500/μL from transplantation, will be analyzed by the Kaplan-Meier method.
Engraftment failure is defined as not achieving neutrophil engraftment by Day 42 post-CTX001
infusion or receipt of backup stem cells. The number and percentage of subjects with
engraftment failure will be summarized.
Time to platelet engraftment, defined as first of 3 consecutive measurements on 3 separate days
with platelet ≥50 × 109/L without a platelet transfusion for 7 consecutive days, will be assessed
by the Kaplan-Meier method.
Laboratory abnormalities (values outside of normal ranges, and by Common Terminology
Criteria for Adverse Events [CTCAE] grade), will also be tabulated.
The number and percentage of subjects with TRM within 100 days and 1 year post CTX001
infusion will be summarized, where TRM is defined as death at least possibly related to the
transplant-related.
12.3.5 Engraftment
Neutrophil engraftment is defined as the first day of 3 measurements of ANC ≥500/µL, achieved
within 42 days post CTX001 infusion, without use of the unmodified CD34+ cells after reaching
the nadir, defined as ANC <500/µL.
Platelet engraftment is defined as the first of 3 consecutive measurements on 3 separate days
with platelet ≥50 × 109/L without a platelet transfusion for 7 consecutive days.
The number and percentage of subjects achieving successful engraftment, together with the
95% confidence interval will be provided.
Time to neutrophil engraftment will be analyzed by the Kaplan-Meier method. Engraftment
failure is defined as not achieving neutrophil engraftment by Day 42 after CTX001 infusion, or
receipt of backup stem cells. The number and percentage of subjects with neutrophil engraftment
failure will be summarized.
Time to platelet engraftment will be analyzed by the Kaplan-Meier method.
AEs will be coded according to MedDRA. AE and SAE summaries will be presented by
MedDRA System Organ Class and Preferred Term using frequency counts and percentages
(i.e., number and percentage of subjects with an event). When summarizing the number and
percentage of subjects with an event, subjects with multiple occurrences of the same AE (SAE)
or a continuing AE (SAE) will be counted once. Only the maximum severity level will be

presented in the severity summaries, and the strongest relationship level will be presented in the
relationship summaries.
 AEs by strongest relationship
 AEs by maximum severity
 SAEs
 AEs leading to death
Details for imputing missing or partial start dates of AEs will be specified in the SAP.
12.3.6.1 Clinical Laboratory Assessments
hematology and chemistry results, including coagulation studies will be summarized in SI units
by visit.
12.3.6.2 Electrocardiogram
will be provided by visit for the following standard 12-lead ECG measurements: RR (msec), HR
HR intervals (QTcF [msec]).
12.3.6.3 Vital Signs
12.3.6.4 Physical Examination
PE findings will be presented in individual subject data listings only.
12.3.7 Exploratory Analysis
Descriptive statistics will be used to describe the following exploratory endpoints (unless stated
otherwise):
 Change in hemolytic index as measured by principal component analysis of 4 markers of
hemolysis (reticulocyte count, serum concentrations of AST, LDH, and total bilirubin) will
 Change in TRV will be summarized as a continuous variable over time.
 Change in inflammatory markers will be summarized as a continuous variable over time.
 Change in endothelial activation markers will be summarized as a continuous variable over
time.

 Change in proportion of circulating erythrocytes expressing fetal hemoglobin (F-cells)

(pre-transfusion) over time
 CD34+ counts
 Correlations of response markers (transfusions, total and fetal hemoglobin) with
pre-treatment variables (e.g., subject genotype), cell dose and percent edited cells in final
product.
clinical study report.
12.3.8 Interim and Independent Data Monitoring Committee Analyses
12.3.8.1 Interim Analysis
12.3.8.2 Independent Data Monitoring Committee Analysis

The DMC review will include review of safety and efficacy data. Details will be included in the
DMC charter and DMC SAP.
13 PROCEDURAL, ETHICAL, REGULATORY, AND ADMINISTRATIVE

CONSIDERATIONS
13.1 Adverse Event and Serious Adverse Event Documentation, Severity

Grading, and Reporting
13.1.1.1 Definition of an Adverse Event


frequency) after the ICF is signed. A pre-existing condition that is diagnosed prior to subject
signing ICF should be recorded in medical history.
An AE is considered serious if it meets the definition in Section 13.1.2.1.
13.1.1.2 Clinically Significant Assessments
Study assessments including laboratory tests, ECGs, PEs, and vital signs will be assessed and
those deemed to have clinically significant worsening from baseline will be documented as an
AE. When possible, a clinical diagnosis for the study assessment will be provided, rather than the
abnormal test result alone (e.g., urinary tract infection, anemia). In the absence of a diagnosis,
the abnormal study assessment itself will be listed as the AE (e.g., bacteria in urine or decreased
hemoglobin).
the following:
 Concomitant signs or symptoms related to the abnormal study assessment
 Further diagnostic testing or medical/surgical intervention
 Discontinuation from the study
A laboratory value that is Grade 4 will not automatically be an SAE. A Grade 4 laboratory value
will be an SAE if the subject’s clinical status indicates a life-threatening AE.
context, Grade 1 and 2 laboratory abnormalities reflective of and fully explained by busulfan
13.1.1.3 Documentation of Adverse Events
AEs will be monitored and collected as outlined in Table 13-1.

transfusion regimen, and excluding
AEs possibly related to fertility
preservation procedures)
mobilization cycle


start of CTX001 infusion
Start of CTX001 infusion to All AEs All SAEs
Month 24 Visit (end of study visit)
 Description of the event
 Classification of “serious” or “nonserious”
 Date of first occurrence and date of resolution (if applicable)
 Severity
 Causal relationship to study drug(s)
 Action taken
 Outcome
 Concomitant medication or other treatment given
13.1.1.4 Adverse Event Severity
guidance available at the following website will be consulted: CTCAE, Version 5.0, Cancer
https://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm#ctc_50 (accessed
November 2018). The severity of an AE that does not appear in the CTCAE will be determined
according to the definitions in Table 13-2.

Mild (Grade 1) Mild level of discomfort and does not interfere with regular activities
Moderate (Grade 2) Moderate level of discomfort and significantly interferes with regular activities
Severe (Grade 3) Significant level of discomfort and prevents regular activities
Life-threatening Any adverse drug event that places the subject, in the view of the investigator, at
(Grade 4) immediate risk of death
Death (Grade 5) Any adverse event that results in the death of the subject


13.1.1.5 Adverse Event Causality

study drug(s). Causality will be classified using the categories in Table 13-3.
Table 13-3 Classifications for AE Causality

Related There is an association between the event and the administration of investigational
study drug, a plausible mechanism for the event to be related to the investigational
study drug and causes other than the investigational study drug have been ruled out,
and/or the event reappeared on re-exposure to the investigational study drug.
investigational study drug and there is a plausible mechanism for the event to be
related to investigational study drug, but there may also be alternative etiology,
such as characteristics of the subject’s clinical status or underlying disease.
Unlikely related The event is unlikely to be related to the investigational study drug and likely to be
related to factors other than investigational study drug.
Not related The event is related to an etiology other than the investigational study drug (the
alternative etiology will be documented in the subject’s medical record).
13.1.1.6 Study Drug Action Taken

The investigator will classify the study drug action taken with regard to the AE. The action taken
will be classified according to the categories in Table 13-4.
Table 13-4 Classifications for Study Drug Action Taken With Regard to an AE
Dose not changed Study drug dose not changed in response to an AE
Dose reduced Study drug dose reduced in response to an AE
Drug interrupted Study drug administration interrupted in response to an AE
Drug withdrawn Study drug administration permanently discontinued in response to an AE
Not applicable Action taken regarding study drug administration does not apply.
“Not applicable” will be used in circumstances such as when the investigational
treatment had been completed before the AE began and no opportunity to decide
whether to continue, interrupt, or withdraw treatment is possible.
13.1.1.7 Adverse Event Outcome

The outcome will be classified according to the categories in Table 13-5.

Table 13-5 Classifications for Outcome of an AE

Recovered/resolved Resolution of an AE with no residual signs or symptoms
Recovered/resolved with Resolution of an AE with residual signs or symptoms
sequelae
Not recovered/not Either incomplete improvement or no improvement of an AE, such that it remains
resolved (continuing) ongoing
Fatal Outcome of an AE is death. “Fatal” will be used when death is at least possibly
related to the AE.
13.1.1.8 Treatment Given

The investigator ensures adequate medical care is provided to subjects for any AEs, including
clinically significant laboratory values related to study drug. In addition, the investigator will
describe whether any treatment was given for the AE. “Yes” is used if any treatment was given
in response to an AE, and may include treatments such as other medications, surgery, or physical
therapy. “No” indicates the absence of any kind of treatment for an AE.
13.1.2 Serious Adverse Events
13.1.2.1 Definition of a Serious Adverse Event
 Fatal (death, regardless of cause, that occurs during participation in the study or occurs after
participation and is suspected of being a delayed toxicity due to administration of the study
drug)
 Life-threatening, such that the subject was at immediate risk of death from the reaction as it
occurred
 Inpatient hospitalization or prolongation of hospitalization. Please note that hospital
mobilization/apheresis, central line placement, busulfan conditioning, CTX001 infusion) do
not meet this criterion.
 Persistent or significant disability/incapacity (disability is defined as a substantial disruption
of a person’s ability to conduct normal life functions)
 Congenital anomaly or birth defect
 Important medical event that, based upon appropriate medical judgment, may jeopardize the
subject or may require medical or surgical intervention to prevent 1 of the outcomes listed
above (e.g., an allergic bronchospasm requiring intensive treatment in an emergency room or
at home)
 Engraftment failure: failure to achieve neutrophil engraftment by Day 42 after CTX001
infusion or need to receive backup stem cells at any time during period of neutropenia
 Development of new malignancy

“serious”, which is based on subject/event outcome or action described above, and is usually
associated with events that pose a threat to a subject’s life or functioning. Seriousness, not
13.1.2.2 Reporting and Documentation of Serious Adverse Events
Month 24 Visit after infusion of CTX001, regardless of causality, will be reported by the
investigator to Vertex GPS within 24 hours of identification. In addition, all SAEs that occur
after the 24-Month Visit and are considered related to study drug(s) will be reported to Vertex
GPS within 24 hours of identification.
Fax: +1-617-341-6159
In parallel with reporting via the SAE Form described above, the investigator must also
immediately notify the medical monitor of any of the following:


 Malignancies
 Grade 4 VOCs associated with mobilization (see Section 9.9.1)
13.1.2.3 Expedited Reporting and Investigator Safety Letters
The sponsor is responsible for reporting suspected, unexpected, serious adverse reactions
(SUSARs) involving the study drug(s) to all regulatory authorities, IECs, and participating
investigators in accordance with ICH Guidelines and/or local regulatory requirements, as
applicable. In addition, the sponsor, or authorized designee, will be responsible for the
submission of safety letters to central IECs.
It is the responsibility of the investigator or designee to promptly notify the local IRB/IEC of all
unexpected serious adverse drug reactions involving risk to human subjects. Investigators will
also be notified of all unexpected, serious, drug-related events (7/15 Day Safety Reports) that
occur during the study. Each site is responsible for notifying its IRB or EC of these additional
SAEs.
13.2 Administrative Requirements
13.2.1 Ethical Considerations
The study will be conducted in accordance with the current ICH GCP Guidelines, which are
consistent with the ethical principles founded in the Declaration of Helsinki, and in accordance
with local applicable laws and regulations. The IRB/IEC will review all appropriate study
documentation to safeguard the rights, safety, and well-being of the subjects. The study will be
conducted only at sites where IRB/IEC approval has been obtained. The protocol, Investigator’s
Brochure, sample ICF, advertisements (if applicable), written information given to the subjects
(including diary cards), safety updates, annual progress reports, and any revisions to these
documents will be provided to the IRB/IEC by the investigator or Sponsor, as allowable by local
applicable laws and regulations.
Ethics Committee/Institutional Review Board
Ethics Review
subjects.

13.2.2 Subject Information and Informed Consent
After the study has been fully explained, written informed consent will be obtained from the
subject or legal representative or guardian (if applicable), and assent will be obtained from the
subject (where applicable), before study participation. The method of obtaining and documenting
the informed consent and the contents of the consent will comply with ICH GCP and all
applicable laws and regulations and will be subject to approval by the sponsor or its designee.
13.2.3 Investigator Compliance
No modifications to the protocol will be made without the approval of both the investigator and
the Sponsor. Changes that significantly affect the safety of the subjects, the scope of the
investigation, or the scientific quality of the study (i.e., efficacy assessments) will require
IRB/IEC notification before implementation, except where the modification is necessary to
eliminate an apparent immediate hazard to human subjects. The Sponsor will submit all protocol
modifications to the required regulatory authorities.
When circumstances require an immediate departure from procedures set forth in the protocol,
the investigator will contact the Sponsor to discuss the planned course of action. If possible,
contact will be made before the implementation of any changes. Any departures from the
protocol will be fully documented in the source documentation and in a protocol deviation log.
13.2.4 Access to Records
The investigator will make the office and/or hospital records of subjects enrolled in this study
available for inspection by the Sponsor or its representative at the time of each monitoring visit
and for audits. The records will also be available for direct inspection, verification, and copying,
as required by applicable laws and regulations, by officials of the regulatory health authorities
(FDA and others). The investigator will comply with applicable privacy and security laws for use
and disclosure of information related to the research set forth in this protocol.
13.2.5 Subject Privacy
laws and regulations, all data provided to the Sponsor, study reports, and communications
relating to the study will identify subjects by assigned subject numbers, and access to subject
names linked to such numbers will be limited to the site and the study physician and will not be
disclosed to the Sponsor. As required by applicable laws and regulations in the countries in
which the study is being conducted, the investigator will allow Sponsor and/or its representatives
access to all pertinent medical records to allow for the verification of data gathered and the
review of the data collection process. The FDA and regulatory authorities in other jurisdictions,
including the IRB/IEC, may also request access to all study records, including source
documentation, for inspection.
For sites participating in the US, and in accordance with the Health Insurance Portability and
Accountability Act (HIPAA) and associated regulations, an executed HIPAA authorization will
be obtained by the site from each subject (or the legal representative of the subject) before
research activities may begin. Each HIPAA authorization will comply with all HIPAA

requirements including authorization allowing the site access to and use of the subject’s
personally identifiable health information, authorization for the site to disclose such information
to Sponsor, the FDA, and other parties requiring access under the protocol, and statements as to
the purpose for which such information may be used and for how long.
13.2.6 Record Retention
The investigator will maintain all study records according to ICH GCP Guidelines and/or
applicable local regulatory requirement(s), whichever is longest, as described in the Clinical
Trial Agreement. If the investigator withdraws from the responsibility of keeping the study
records, custody will be transferred to a person willing to accept the responsibility and the
Sponsor will be notified.
13.2.7 Study Termination
to:
 Subject or investigator noncompliance
 Unsatisfactory subject enrollment
 Lack of adherence to protocol procedures
 Lack of evaluable and/or complete data
 Potentially unacceptable risk to study subjects
 Decision to modify drug development plan
 Decision by the FDA or other regulatory authority
All subjects who receive CTX001 will be asked to enroll in Study VX18-CTX001-131, a
long-term follow-up study, to be followed for a total of up to 15 years after infusion.
13.2.8 End of Study
End of study will be defined as the date the last subject completes the 24-month follow-up period
or date of early withdrawal from the study. The Sponsor will notify all applicable regulatory
agencies in accordance with local requirements when the study has ended.

13.3 Data Quality Assurance

Sponsor or its designated representative will conduct a study site visit to verify the qualifications
of each investigator, inspect clinical study site facilities, and inform the investigator of
responsibilities and procedures for ensuring adequate and correct study documentation.
The investigator is required to prepare and maintain adequate and accurate case histories
designed to record all observations and other data pertinent to the study for each subject. Study
data for each enrolled subject will be entered into a CRF by study site personnel using a secure,
validated, web-based electronic data capture (EDC) application. The Sponsor will have read-only
access to site-entered clinical data in the EDC application.
Instances of missing, discrepant, or uninterpretable data will be queried with the investigator for
resolution. Any changes to study data will be made to the CRF and documented in an audit trail,
which will be maintained within the clinical database.
13.4 Monitoring
 Determine the adequacy of the facilities
 Discuss with the investigator(s) and other personnel their responsibilities with regard to
comply with GCP Guidelines. On-site checking of the CRFs/SAE Forms for completeness and
clarity, cross-checking with source documents, and clarification of administrative matters will be
performed.
The study will be monitored by the Sponsor or its designee. Monitoring will be done by personal
 Provide information and support to the investigator(s)
 Confirm that facilities remain acceptable
 Confirm that the investigational team is adhering to the protocol, that data is being
are being performed

13.5 Electronic Data Capture

A CRF will be completed for each consented study subject. It is the investigator’s responsibility
to ensure the accuracy, completeness, clarity, and timeliness of the data reported in the subject’s
CRF. Source documentation supporting the CRF data will indicate the subject’s participation in
and subject status.
The audit trail entry will show the user’s identification information and the date and time of any
including any changes made to them, to endorse the final submitted data for the subjects for
CRF in the form of a compact disc (CD) or other electronic media will be placed in the
investigator’s study file.
13.6 Publications and Clinical Study Report
13.6.1 Publication of Study Results
information to any third party except to such of the investigator’s employees and staff as have
if the parties decide to proceed with it, for the purpose of conducting the study.
partners and associates, the FDA, and other government agencies. The investigator also
understands that, to allow for the use of the information derived from the study, the investigator
has the obligation to provide the Sponsor with complete test results and all data developed in the
study.
conditions of a separate written agreement between the Sponsor and the investigator and/or the
investigator’s institution.
13.6.2 Clinical Study Report
A clinical study report, written in accordance with the ICH E3 Guideline, will be submitted in
accordance with local regulations.

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15.2 Investigator Signature Page

Protocol #: CTX001-121 Version #: 5.0 Version 04 February 2020
Date:
Study Title: A Phase 1/2 Study to Evaluate the Safety and Efficacy of a Single Dose of
Autologous CRISPR-Cas9 Modified CD34+ Human Hematopoietic Stem and Progenitor Cells
(CTX001) in Subjects With Severe Sickle Cell Disease
I have read Protocol CTX001-121, Version 5.0, and agree to conduct the study according to its
terms. I understand that all information concerning CTX001 and this protocol supplied to me is
confidential.
Printed Name
Signature Date

16 APPENDICES
16.1 Busulfan PK Collection

Samples for PK analysis of busulfan will be drawn on the 1st and 3rd day of dosing using
at the following time points.
End of infusion, (± 5 minutes)
End of infusion + 15 minutes (± 5 minutes)
Start of infusion + 4 hours (± 15 minutes)
Recommendations for dosing q6h: Samples for busulfan PK analysis should be collected at the
following time points.
End of infusion (± 5 minutes)
above.
adjustments will be included in the electronic case report form (eCRF).

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Protocol

Clinical Study Protocol CTX001-111

Clinical Study Protocol CTX001-121

1. Original protocol (Version 1.0) – p183

PROTOCOL ACCEPTANCE FORM

Date: November 22, 2017

Investigator’s Signature Date

Version 1.0 3 Confidential

Version 1.0 4 Confidential

Version 1.0 5 Confidential

Version 1.0 6 Confidential

Version 1.0 7 Confidential

Version 1.0 8 Confidential

TABLE OF CONTENTS, LIST OF TABLES, AND LIST OF FIGURES

Version 1.0 9 Confidential

3.5. Waivers of Inclusion/Exclusion Criteria .........................................................................33

Version 1.0 10 Confidential

8.8. Laboratory Assessments ..................................................................................................55

Version 1.0 11 Confidential

11.2. Electronic Data Capture (EDC) ..................................................................................71

Version 1.0 12 Confidential

Version 1.0 13 Confidential

LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

Abbreviation or Specialist Term Explanation

Version 1.0 14 Confidential

Abbreviation or Specialist Term Explanation

Version 1.0 15 Confidential

Abbreviation or Specialist Term Explanation

Version 1.0 16 Confidential

Abbreviation or Specialist Term Explanation

Version 1.0 17 Confidential

Figure 1 Molecular Structure of Hemoglobin A (OpenStax College, 2016)

Version 1.0 18 Confidential

1.2. Current Treatment Approaches for Transfusion-Dependent

Version 1.0 20 Confidential

1.3. CRISPR-Cas9 Gene Editing Approach

Figure 3 Schematic of the CRISPR-Cas9 Complex

Version 1.0 21 Confidential

Figure 4 CRISPR-Cas9-Mediated Genome Editing Strategies

Version 1.0 22 Confidential

1.5. Investigational Product: CTX001

Version 1.0 23 Confidential

1.10. Risk Assessment

1.10.1. Potential Risks of CTX001

Version 1.0 27 Confidential

1.10.2. Potential Risks of Mobilization, Apheresis and Autologous Transplantation

Version 1.0 28 Confidential

2. STUDY OBJECTIVES AND ENDPOINTS

Version 1.0 29 Confidential

2.2.2. Primary Efficacy Endpoint

Version 1.0 30 Confidential

3.1. Number of Subjects

3.2. Subject Inclusion Criteria

Version 1.0 31 Confidential

3.3. Subject Exclusion Criteria

Version 1.0 32 Confidential

3.4. Enrolled Subjects and Screen Failures

3.5. Waivers of Inclusion/Exclusion Criteria

Version 1.0 33 Confidential

Version 1.0 34 Confidential

3.8. Subject Withdrawal

3.9. Replacement of Subjects

Version 1.0 36 Confidential