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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