Focus Biosciences I - HP-F10 : Epigenomics & Cancer

Highly efficient therapeutic gene editing

of human hematopoietic stem cells
Yuxuan Wu, Jing Zeng,… Scot A. Wolfe5 and Daniel E. Bauer 1,2,3*

Supervisors:
Dr. Ashish Goyal
Dr. Maria Llamazares Prada

Toros Taşgın
Master Programme “Molecular Biosciences“, Major “Cancer Biology“ University of Heidelberg and DKFZ

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 Introduction: Hemoglobin (Hb)

• Tetrameric protein composed of two ⍺-like globin chains and

two β-like globin chains.

• Human β-like globin locus contains five β-like globin genes (ε, δ,
β, Aγ, and Gγ).

• ε-globin is expressed during the embryonic stage, then replaced

by γ-globin during fetal life. Around the time of birth, γ to β
globin switch occurs.1

• The induction of HbA is regulated by a pentameric complex, and

BCL11A protein is the major determinant for γ-globin
repression.2-7

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Introduction: β-hemoglobinopathies
• Sickle cell disease (SCD) and β-Thalassemia are most common monogenic disorders
worldwide, with approximately 300.000 affected neonates born each year. 8

β-Thalassemia;

• Caused by more than 200 different β-globin gene mutations.

• Reduced or abolished β-globin expression.
• Excess ⍺-globin precipitates and leads ineffective
erythropoiesis, apoptosis of erythroid precursors, and
hemolytic anemia.9

SCD;

• Caused by A to T mutation in the sixth codon of β-globin (E6V).

• Defected β-globins (βS) form protein aggregates (HbS polymer) in
deoxygenated state.
• Protein aggregates lead deformation of erythroid cell morphology
(sickle/hook).
• Blockage of microvessels and cause multi-organ impairments. 9

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Therapeutic Strategy
β-Thalassemia: Reduction of ⍺-β
globin imbalance=prevention of ⍺
globin precipitation
SCD: Inhibition of HbS
polymerization

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Characterization of single guide RNAs (sgRNAs)

RNP complex Sanger

(Cas9+sgRNA) TIDE analysis = editing efficiency
sequencing

RT-qPCR = γ-globin and BCL11A expression

RNP In vitro
electroporation HPLC = HbF protein level
+ + differentiation
CD34 HSPCs Edited CD34
Edited
from healthy HSPCs Erythrocytes Flow Cytometry= HbF+ erythrocytes (%)
donor

TIDE analysis RT-qPCR HPLC Flow Cytometry

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 Clonal Analysis of Single Origin Erythrocytes

Genotyping
RT-qPCR = γ-globin expression
Cell sorting
Cas9+sgRNA-1617
In vitro differentiation
(11 days)
Edited CD34+ HPLC = % HbF protein
HSPC cells from
healthy donor

RT-qPCR HPLC
Edited
erythrocytes

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γ-globin Induction in Patient HSPCs
Sanger TIDE analysis = editing efficiency
sequencing

Cas9+sgRNA-1617 RT-qPCR = γ-globin expression

In vitro differentiation
Edited
Edited CD34+ Erythrocytes
Cas9+sgRNA-AAVS1 HSPC cells from
HPLC = % of HbF protein
different patients

(normalized by ⍺-globin)

Healthy
β-thalassemia

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Effect of Enhancer editing on Erythroid maturation

% of Hoechst
negative cells
Cas9+sgRNA-1617 Edited cells from
different patients Flow
In vitro Nuclear Cytometry Cell size
differentiation Staining
Cas9+sgRNA-AAVS1 (Hoechst 33342)
Edited cells from Circularity Analysis
healthy donor

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Permanent Modification of Stem Cells
TIDE analysis
(prevalence of edited human cells)

Human B cell
Cell progeny
Engraftment FACS
BM isolation
BCL11A
RT-qPCR
Cas9+sgRNA-1617 expression
NBSGW mice (16-weeks post-engraftment)
(supports HSPC engraftment) Human
+
Edited CD34 Erythroid
HSPC cells from progeny
healthy donors
RT-qPCR
!!! Lineage specific activity of +58
BCL11A enhancer γ-globin
expression

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Improvement of Editing Efficiency

2xNLS-Cas9 3xNLS-Cas9
3xNLS-Cas9+sgRNA-1617
Addition of c-Myc-like NLS Edited CD34+
HSPC cells

TIDE analysis
Amplicon deep sequencing

Quantification of editing frequency via

Amplicon deep sequencing

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Elevated levels of γ-globin and HbF

RT-qPCR = γ-globin expression

In vitro differentiation
17 days
3xNLS-Cas9+sgRNA-1617 HPLC = % HbF protein
Edited CD34+
5 7 9 11 13 15
HSPC cells

RT-qPCR Western blot + image analysis

(BCL11A expression) (BCL11A protein levels)

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Enhanced Therapeutic Editing TIDE analysis
(prevalence of edited human cells)

Human B cell
Cell progeny
Engraftment FACS
BM isolation
BCL11A
RT-qPCR
3xNLS-Cas9+sgRNA-1617 expression
NBSGW mice (16-weeks post-engraftment)
(supports HSPC engraftment) Human
+
Edited CD34 Erythroid
HSPC cells from progeny
healthy donors
RT-qPCR

γ-globin
expression

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Effects of Editing in SCD

MBS treatment

NBSGW mice Edited CD34+ Edited Resistant edited

(transplanted with edited erythrocytes erythrocytes
HSPC cells from
HSPCs from SCD donor )
SCD donor In vitro differentiation

BM isolation + FACS MBS treatment

(16-weeks post-engraftment)
NBSGW mice Unedited
Unedited CD34+ Sickled unedited
(transplanted with unedited erythrocytes
HSPC cells from erythrocytes
HSPCs from SCD donor ) SCD donor

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Summary

• Development of selection-free, HSC expansion-free BCL11A enhancer editing

strategy.

• Improvement of editing efficiency by Cas9 protein with additional NLS.

• Persistent indel frequency in long-term engraftments comparable to input

indel frequency.

• Overall, improved therapeutic efficacy.

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1. Sankaran, V.G., and Orkin, S.H. (2013). The switch from fetal to adult hemoglobin. Cold Spring Harb. Perspect. Med. 3,
a011643.
2. Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel, B., Mikkola, H.K., Hirschhorn, J.N., Cantor, A.B., and
Orkin, S.H. (2008). Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
Science 322, 1839–1842.
3. Bauer, D. E. et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science
342, 253–257 (2013).
4. Canver, M. C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197
(2015).
5. Smith, E. et al. Strict in vivo specificity of the Bcl11a erythroid enhancer. Blood 128, 2338–2342 (2016).
6. Vierstra, J. et al. Functional footprinting of regulatory DNA. Nat. Methods 12, 927–930 (2015).
7. Chang, K.-H. et al. Long-term engraftment and fetal globin induction upon BCL11A gene editing in bone-marrow derived
CD34+ hematopoietic stem and progenitor cells. Mol. Ther. Methods Clin. Dev. 4, 137–148 (2017).
8. Sankaran, V.G., Menne, T.F., Xu, J., Akie, T.E., Lettre, G., Van Handel, B., Mikkola, H.K., Hirschhorn, J.N., Cantor, A.B., and
Orkin, S.H. (2008). Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A.
Science 322, 1839–1842.
9. Marina Cavazzana, Chiara Antoniani, Annarita Miccio, Gene Therapy for β-Hemoglobinopathies,Molecular Therapy, Volume
25, Issue 5, 2017, Pages 1142-1154, ISSN 1525-0016, https://doi.org/10.1016/j.ymthe.2017.03.024.

Figures were adapted from the paper in reference “9“ and generated via Biorender

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 20-off target sites

Amplicon deep sequencing

20-off target sites: no 80-95% editing freq in

edited sequence BCL11A enhancer

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 Permanent Modification of Stem Cells

+¿
% of human 45
%h𝐶𝐷
  +¿ ¿
WB cells % h𝐶𝐷45+¿+%𝑚𝐶𝐷 45 ¿ ¿
Cell
Engraftment Flow
BM isolation Cytometry TIDE analysis
Cas9+sgRNA-1617 (16-weeks post-engraftment) (prevalence of edited human cells)
NBSGW mice
(supports HSPC engraftment)
Edited CD34+
HSPC cells from % of human 235 𝑎+¿
%h𝐶𝐷
healthy donors erythroid cells   −
% h𝐶𝐷 45 +%𝑚𝐶𝐷 45
−
¿

0.4 million cells 0.8 million cells

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Enhanced Indel Frequency and HbF induction

Multilineage reconstitution

Cell
Engraftment Flow % of human cells
BM isolation Cytometry
3xNLS-Cas9+sgRNA-1617 TIDE analysis
NBSGW mice (16-weeks post-engraftment)
(supports HSPC engraftment)
Edited CD34+
HSPC cells from
healthy donors

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Effects of Editing in SCD

γ-globin
RT-qPCR
TIDE analysis expression

tio n
d i fferentia
o
In vitr
3xNLS-Cas9+sgRNA-1617

γ-globin
RT-qPCR
Clonal analysis expression
Edited CD34+
3xNLS-Cas9+AAVS1
HSPC cells from
SCD donor
Genotyping

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 Effects of Editing in SCD

Flow % of human cells TIDE analysis

Cytometry

Cell
Engraftment
BM isolation
BCL11A
FACS RT-qPCR
3xNLS-Cas9+sgRNA-1617 expression
NBSGW mice (16-weeks post-engraftment) (separation of B cells and
(supports HSPC engraftment) erythrocytes)
Edited CD34+ γ-globin
HSPC cells from expression
different SCD
donors

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 Effects of Editing in SCD

TIDE analysis
Cell
Engraftment
BM isolation
BCL11A
FACS RT-qPCR
3xNLS-Cas9+sgRNA-1617 expression
NBSGW mice (16-weeks post-engraftment) (separation of B cells and
(supports HSPC engraftment) erythrocytes)
Edited CD34+ γ-globin
HSPC cells from expression
different SCD
donors

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