1. 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 23 rd November 2008Thalea Koithan

2. HIV life cycle 2 University of Washington (2004)

3. CXCR4 and CCR5 coreceptors CXCR4CCR5 T cell-tropic HIVmacrophage-tropic HIV syncytium-inducing (SI) HIV-1non-syncytiuminducing (NSI) HIV-1 appears during later stagespredominates during early infection associated with progression to AIDS responsible for viral transmission HIV-1, HIV-2HIV-1, HIV-2, SIV 3 dual-tropic HIV strains

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

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

6. ZFN-mediated disruption of CCR5 6 FokI cleavage NHEJ Surveyor Assay 2. Digest re-annealed products and analyze by PAGE. 1.Denature and allow PCR products to re-anneal to wildtype template.

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

8. ZFN-mediated disruption of CCR5 8 CCR5 ZFNs-transduced GHOST cells After 1 week Reduced CCR5 surface expression (>10-fold) Decreased infection with CCR5- tropic HIV-1 Unstained cells IL2R γ ZFN CCR5 ZFN-224 CCR5 ZFN-215 Nontransduced cells IL2R γ ZFN CCR5 ZFN-224 CCR5 ZFN-215

9. Resistance of CCR5 ZFN-treated GHOST-CCR5 cell clones to HIV infection 9 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

10. In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4 + T cell line PM1 10 PM1 = CD4 + T cell line, similar levels of CCR5 expression to primary CD4 + T cells

11. In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4 + T cell line PM1 11 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

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

13. 13 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-transduced ZFN-224 GFP-transduced Infection with CCR5-tropiv HIV-1 2x enrichment of gene-edited cells with ZFN-disrupted CCR5 alleles HIV-1 infected mock infected

14. 14 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 NHEJ Quantification of ZFN action throughout the nucleus

15. 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 16 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. 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%) 17 Determination of the consensus binding site for CCR5-ZFN

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

19. 19 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

20. 50 days after infection 8 of 10 HIV-infected mice >50% CCR5-disrupted CD4 + T cells in peripheral blood 20 Reduction in viremia and selection for CCR5 ZFN-modified CD4 + T cells in the presence of HIV-1 challenge in vivo

21. 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-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

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 22

23. CCR5 versus CXCR4 23

24. ZFN 24

25. 25

26. 26