Newer Gene Editing Technologies toward HIV Gene

Therapy

N. Manjunath, Guohua Yi, Y. Dang, P. Shankar

BTEC 643
Ezgi Tanl
Overview
Introduction to HIV therapies
ZFNs and their use in HIV gene therapy
Specificity and delivery methods for ZFN
TALENs and their use in HIV gene therapy
CRISPR/Cas9 system and its use in HIV gene
therapy
1. Introduction
HIV
Become manageable by using anti-retroviral
therapies
HAART : highly active anti-retroviral therapy
Suppress viral replication
Limitations :
High cost
Patient compliance
Side effects of long term therapy
Emergence of drug resistance
1. Introduction
CCR5:
One of the major co-receptors for HIV
Berlin patient:
HIV(+) lenfoma patient
Transplanted with bone marrow from a CCR5-32
homozygous donor
Limitations:
Low frequency of CCR5-32 homozygous donor
Difficulties of identifying suitable donors
Alternative methods should be developed
1. Introduction
RNA interference
Requires continuous presence of small interfering
RNA (siRNA)
Gene ablation is never complete
Targeted gene disruption techniques are developed
General strategy:
Double strand breaks by nucleases
Repair of the nick by:
Homologous recombination
Error-prone nonhomologous end joining (NHEJ)
2. Zinc-Finger Nucleases
Zinc-Finger proteins
recognizes nucleotide triplets.

Fok-1 nucleases perform

double strand break

Stimulation of cellular DNA

repair mechanisms
Homologous
recombination
Nonhomologous end
joining
 2. Zinc-Finger Nucleases
HIV-1 treatment:
Conventional RNAi
silencing
Co-receptor
CCR5/CXC4
disruption
Targeting HIV-1
genome
Gene disruption
methods are heritable
Image: https://www.researchgate.net/publication/221811209/figure/fig5/AS:203147638251550@1425445579994/Chemokines-and-their-receptors-network-guiding-the-HIV-entry-CCR5-
and-CXCR4-co-receptor.png
2.1. CCR5 Disruption
CD4 :
Major viral receptor
Important roles in immune system
CCR5 :
No major immune deficits
Predominant in early stages of HIV entry
Target to block HIV-1 entry
2.1. CCR5 Disruption
Mani et al 2005:
First report on CCR5 alteration by ZFN technology
Cleavage of CCR5 using three finger ZFN
CCR5 disruption performed on
Primary T cells
Hematopoietic stem cells (HSCs)
Humanized mice
2.1.1. CCR5 disruption in CD4+ T cells
Perez et al. (2008):
CCR5 was disrupted in 50% of the cells.
Significant resistance to HIV-1
T cells are easily enriched
Early results of clinical trials:
Harmless persistent T cells obtained
The cells traffic to different organs
2.1.2 CCR5 disruption in HSCs
Yao et al (2012):
Disruption of CCR5 in embriyonic and induced pluripotent
stem cells
Holt et al (2010):
Nucleofection to achieve ZFN mediated CCR5 disruption in
human CD34+ HSCs
Lombardo et al (2007):
Disruption of CCR5 using non-integrating lentiviral vector-
delivered ZFN in HSCs
Li et al (2013):
Disruption of CCR5 using adenoviral vector delivered ZFN.
2.2. CXCR4 disruption
CXCR4:
Can be chosen as an alternative after infection is established
CXCR4 tropic viruses emerge
Wilen et al (2011):
First group to engineer CXCR4 deficient CD4+ T cells by CXCR4-ZFN delivery
via AD5/35
Resistance to X4 HIV-1
Yuan et al (2012):
Comparison of the efficiency of X4 targeting shRNAs & ZFN
ZFN was shown to be superior to shRNA
Disruption of CCR5 or CXCR4 only is not sufficient to provide stable
protection.
Voit et al (2013):
Used ZFN to knock out both CCR5 and CXCR4
Robust resistance to both R5-tropic and X4-tropic HIV-1
2. 3. Targeted HIV-1 Proviral DNA Disruption
CCR5 and CXCR4 disruption is not sufficient to
eradicate the virus from already infected cells
ZFN is used to target proviral DNA in infections
of:
Hepatitis B virus (HBV)
Herpes simplex 2 virus (HSV-2)
Human T-Cell Lymphotropic Virus -1
2. 3. Targeted HIV-1 Proviral DNA Disruption
Wayengera (2011):
Found ZFN pairs that could delete 80% of proviral DNA.
Das et al (2012):
Designed and screened 3 ZFNs that efficiently cleave conserved
regions of gag, pol and rev genes.
Qu et al (2013):
Designed ZFN pairs to eradicate HIV-1 proviral DNA cell lines.
Limitations:
Patients harbor the integrated HIV-1 provirus in resting T cells
Latently infected cells are extremely rare
Delivery of ZFN to these cells in vivo is difficult
2.4. ZFN delivery strategies for HIV gene
therapy
Mainly focused on CD4+ T cells or CD34+ HSC
Adenoviral vectors:
CD4+ T cells:
54% efficiency for CCR5 and 30-34% efficiency for
CXCR4
CD34+ HSCs:
Efficiency of more than 25% for CCR5
Preexisting immunity may neutralize the virus
2.4. ZFN delivery strategies for HIV gene
therapy
Non-replicating HIV based lentiviral vector
Lombardo et al (2007)
Low efficiency (5%) in CCR5 disruption
Baculoviral vector
Lei et al (2011)
Transduction of hES cells
5% efficiency of CCR5 disruption
2.4. ZFN delivery strategies for HIV gene
therapy
Nonviral methods
Cost effective
Minimal safety concerns
Plasmid transfection by nucleofection
17% CCR5 disruption in HSC
mRNA electroporation
CCR5-ZFN mRNA transfer
Cannon et al (2013)
30-50% efficiency in patient derived HSCs
Persistence after 6 months of reconstitution
2.4. ZFN Delivery Strategies for HIV Gene
Therapy
Gaj et al (2012):
ZFP has intrinsic cell penetrating property.
Incubation of CD4+ T cells with recombinant
CCR5-ZFP
8% efficiency
2.5. Specificity of ZFN targeted gene
disruption in HIV-1 gene therapy
Important for safety concerns
Off-target events should be avoided.
CCR2 :
Homologous locus of CCR5
Kim et al (2009):
Single mismatch between targeted CCR5 sequence and CCR2
gene induce off-target activity
Perez et al (2008):
In the presence of 2 mismatches, CCR5 cleavage is 10 fold higher
Pattanayak et al (2011):
Tight binding of one half can cause off-target effects
ZFN concentration should be as low as possible
3. TALENs
Transcription activator-like
effector nucleases
Naturally occuring DNA
binding proteins from
Xanthomonas
Custom TALE DNA binding
domains fused to Fok-1
endonuclease
Each finger recognizes only
one nucleotide
Variabilities at RVDs (repeat
variable diresidues)
3. TALENs
Assembly of TALENs is a challenge due to high
sequence similarities
REAL (Restriction enzyme and ligation cloning)
High-throughput solid phase ligation strategy
Ligation independent cloning techniques
3. TALENs
Mussolino et al (2001):
Only attempt using the TALEN system for HIV-1
gene therapy
Compared CCR5 gene disruption using ZFN &
TALEN side by side
Both have 45% gene disruption levels
Advantages of TALEN:
Much lower cytotoxicity
Lower off-target activity at the CCR2 locus
3. TALENs
Further studies using humanized mouse models
are necessary
Limitations:
Relatively large size
Expression in lentiviral vectors have failed.
Genome wide off-target effects are not clear
4. Engineered CRISPR/Cas9 System
CRISPR (clustered regularly interspaced
palindromic repeats)
Array of short direct repeats interspersed with
short intervening spacers
Surrounded by Cas genes
Functions in bacteria
Destruction of foreign invaders
Enhancement of virulence
4. Engineered CRISPR/Cas9 System
4. Engineered CRISPR/Cas9 System
Advantages:
Being less labor intensive
Adopts a Watson-Crick complementarity rule
Cas9 is a common moiety already available in cloned form
High versatility
High specificity
Less off-target effects
Online design tools were developed
Dose dependent
No significant cytotoxicity
Double nicking strategy
Single strand nickase can be obtained
Can be repaired by base excision repair
Off target activity decreases 50-150 folds
4. Engineered CRISPR/Cas9 System
Kim et al (2013):
Co-delivery of Cas9 expression construct with
gRNA encoding plasmid
Induced mutations up to 18% at CCR5 locus
Ebina et al (2013):
Used CRISPR/Cas9 system to target HIV-LTR
(long terminal repeat)
5. Conclusion
ZFNs, TALENs and CRISPR/Cas9 are versatile
tools for gene disruption in HIV gene therapy.
The delivery of gene editing mediators needs to
be optimized for relevant cell types
Genome wide off target effects should carefully
be evaluated.
Cytotoxicity of the endonucleases should be
studied.