23 rd November 2008
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Transcript of 23 rd November 2008
Establishment of HIV-1 resistance in CD4+ T cells by genome editing
using zinc-finger nucleases
Elena E Perez, Jianbin Wang, Jeffrey C Miller, Yann Jouvenot, Kenneth A Kim, Olga Liu,Nathaniel Wang, Gary Lee, Victor V Bartsevich, Ya-Li Lee, Dmitry Y Guschin, Igor Rupniewski,
Adam J Waite, Carmine Carpenito, Richard G Carroll, Jordan S Orange, Fyodor D Urnov,Edward J Rebar, Dale Ando, Philip D Gregory, James L Riley, Michael C Holmes & Carl H June
Nature Biotechnology, July 2008
23rd November 2008Thalea Koithan
HIV life cycle
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University of Washington (2004)
CXCR4 and CCR5 coreceptors
CXCR4 CCR5
T cell-tropic HIV macrophage-tropic HIV
syncytium-inducing (SI) HIV-1 non-syncytiuminducing (NSI) HIV-1
appears during later stages predominates during early infection
associated with progression to AIDS
responsible for viral transmission
HIV-1, HIV-2 HIV-1, HIV-2, SIV
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dual-tropic HIV strains
CCR5 ∆32 deletion
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F. Arenzana-Seisdedos, M. Parmentier / Seminars in Immunology 18 (2006) 387–403
ZFNs targeted against CCR5
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Zinc-finger nucleases (ZFNs)
Zinc-finger based DNA binding site DNA cleavage domain Introduce DSB
ZFN-mediated disruption of CCR5
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FokI cleavage NHEJ Surveyor Assay
2. Digest re-annealed products and analyze by PAGE.
1. Denature and allow PCR productsto re-anneal to wildtype template.
ZFN-mediated disruption of CCR5
FokI cleavage NHEJ Surveyor Assay
GHOST-CCR5 cells derived from human
osteosarcoma cells multiple CCR5
expression cassettes Inducible GFP (under
control of HIV-2 LTR)
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CCR5 ZFN-transduced high efficiency target gene mutations
Cleaved products
ZFN-mediated disruption of CCR5
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CCR5 ZFNs-transduced GHOST cells
After 1 week
Reduced CCR5 surface expression (>10-fold)
Decreased infection with CCR5-tropic HIV-1
Unstained cells
IL2Rγ ZFNCCR5 ZFN-224
CCR5 ZFN-215Nontransduced cells
IL2Rγ ZFNCCR5 ZFN-224
CCR5 ZFN-215
Resistance of CCR5 ZFN-treated GHOST-CCR5 cell clones to HIV infection
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Isolation of single cell-derived clones from ZFN-treated GHOST-CCR5 population
Completely resistant to HIV-1 infection
infection of clones with unmodified CCR5 genes
Unstained cells
CCR5 clone
In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T cell line PM1
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PM1 = CD4+ T cell line, similar levels of CCR5 expression to primary CD4+ T cells
In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T cell line PM1
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CCR5 ZFN target region amplified by PCR (day 52 after HIV-1 infection)
Mutations mapped to the core of ZFN recognition site
Permanent modification of CCR5 by ZFN cleavage and repair via NHEJ
Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge
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Transduction of primary human CD4+ T cells
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Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge
Indistinguishable population-doubling rate of modified CD4+ T cells ZFN-224 transduced ( ) nontransduced cells ( ) or control GFP transduced cells ( )
Non-transducedZFN-224GFP-transduced
Infection with CCR5-tropiv HIV-1 2x enrichment of gene-edited cells with ZFN-disrupted CCR5 alleles
HIV-1 infected
mock infected
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Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge
Intranuclear staining for genome-wide DSB via immunodetection of p53 binding protein 1 (53BP1)recruitment of 53BP1 to sites of DSBs early in repair response, required for NHEJQuantification of ZFN action throughout the nucleus
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Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge
Intranuclear 53BP1 foci transiently increased 1.4 – 1.6-fold
Etoposide-treated positive control cells had 4.2-fold increase in 53BP1 staining
Determination of the consensus binding site for CCR5-ZFN
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Production of randomized DNA oligonucleotides that specifically bind to the target ZFN
Sequence alignment for determination of consensus binding site sequence for zinc-finger DNA binding domain
CCR2 is the only off-target with functions in CD4+ T cells ZFN-224 has 10-fold lesser extend of activity at CCR2 (4.1%) than
at CCR5 (35.6%)
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Determination of the consensus binding site for CCR5-ZFN
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Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo
Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo
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Level of ZFN-disrupted CCR5 alleles in CD4+ T cells isolated on day 40 from spleens
3-fold enrichment for ZFN-disrupted CCR5 alleles in HIV-infected group
50 days after infection 8 of 10 HIV-infected mice >50% CCR5-disrupted CD4+ T cells in peripheral blood
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Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo
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Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo
10 days post infection less HIV-1 viral RNA in CCR5 ZFN-treated mice
Engraftment of CD4+ T cells in peripheral blood CCR5 ZFN-treated mice had higher
CD4+ T cell counts on day 30-50 p.i.
GFP
CCR5 ZFN
Summary I
CCR5 ZFNs efficiently cleave their target site in CCR5 HIV-1 infection provides a potent selective advantage for CCR5 ZFN-
modified cells
Modified CD4+ T cells confer resistance to HIV infection in vivo by >50% CCR5-disrupted CD4+ T cells in peripheral blood Increased numbers of CD4+ T cells Lower plasma viremia
Transient delivery of engineered ZFNs could mimic the selective advantage of naturally occurring CCR5∆32 null mutation in humans for resistance to CCR5-tropic HIV-1
Potential to reconstitute immune function by maintainance of an HIV-resistant CD4+ T cell population for clinical trials
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CCR5 versus CXCR4
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ZFN
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