23 rd November 2008

1. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleasesElena 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 JuneNature Biotechnology, July 2008 Thalea Koithan 23rd November 2008

2. HIV life cycle Universityof Washington (2004)

3. CXCR4 and CCR5 coreceptors dual-tropic HIV strains

4. CCR5 ∆32 deletion F. Arenzana-Seisdedos, M. Parmentier / Seminars in Immunology 18 (2006) 387–403

5. ZFNs targeted against CCR5 • Zinc-finger nucleases (ZFNs) • Zinc-finger based DNA binding site • DNA cleavage domain • Introduce DSB

6. ZFN-mediated disruption of CCR5 • FokI cleavage NHEJ Surveyor Assay • Denature and allow PCR products • to re-anneal to wildtype template. 2. Digest re-annealed products and analyze by PAGE.

7. 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) Cleaved products CCR5 ZFN-transduced high efficiency target gene mutations

8. IL2Rγ ZFN CCR5 ZFN-224 IL2Rγ ZFN CCR5 ZFN-224 CCR5 ZFN-215 Nontransduced cells CCR5 ZFN-215 Unstained cells ZFN-mediated disruption of CCR5 • CCR5 ZFNs-transduced GHOST cells • After 1 week • Reduced CCR5 surface expression (>10-fold) • Decreased infection with CCR5-tropic HIV-1

9. Resistance of CCR5 ZFN-treated GHOST-CCR5 cell clones to HIV infection • Isolation ofsinglecell-derivedclonesfrom ZFN-treated GHOST-CCR5 population • Completelyresistantto HIV-1 infection • infectionofcloneswithunmodified CCR5 genes Unstained cells CCR5 clone

10. In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T cell line PM1 • PM1 = CD4+ T cellline, similarlevelsof CCR5 expressiontoprimary CD4+ T cells

11. In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T cell line PM1 • CCR5 ZFN targetregionamplifiedby PCR (day 52 after HIV-1 infection) • Mutationsmappedtothecoreof ZFN recognitionsite • Permanent modificationof CCR5 by ZFN cleavageandrepair via NHEJ

12. Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge • Transductionof primary human CD4+ T cells

13. Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge Non-transduced HIV-1 infected ZFN-224 GFP-transduced mock infected • Indistinguishable population-doubling rate of modified • CD4+ T cells ZFN-224 transduced ( ) nontransduced cells ( ) • or control GFP transduced cells ( ) • Infectionwith CCR5-tropiv HIV-1 • 2x enrichmentofgene-editedcellswith ZFN-disrupted CCR5 alleles

14. Enrichment of CCR5 ZFN-modified primary CD4+ T cells during in vitro HIV-1 challenge • Intranuclearstainingforgenome-wide DSB via immunodetectionof p53 bindingprotein 1 (53BP1) • recruitmentof 53BP1 tositesof DSBs early in repairresponse, requiredfor NHEJ • Quantificationof ZFN actionthroughoutthenucleus

15. 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

16. Determination of the consensus binding site for CCR5-ZFN • 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

17. Determination of the consensus binding site for CCR5-ZFN • 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%)

18. Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo

19. Reduction in viremia and selection for CCR5 ZFN-modified CD4+ T cells in the presence of HIV-1 challenge in vivo • Level of ZFN-disruptedCCR5 alleles in CD4+ T cellsisolated on day 40 fromspleens • 3-fold enrichmentfor ZFN-disruptedCCR5 alleles in HIV-infectedgroup

20. Reduction in viremiaandselectionfor CCR5 ZFN-modified CD4+ T cells in thepresenceof HIV-1 challengein vivo • 50 days after infection 8 of 10 HIV-infected mice >50% CCR5-disrupted CD4+ T cells in peripheral blood

21. 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-treatedmice • Engraftmentof CD4+ T cells in peripheralblood • CCR5 ZFN-treatedmicehadhigher CD4+ T cellcounts on day 30-50 p.i. CCR5 ZFN GFP

22. 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

23. CCR5 versus CXCR4

24. ZFN