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Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases

Nature Biotechnology volume 26, pages 808–816 (2008)Cite this article

Abstract

Homozygosity for the naturally occurring Δ32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted ∼50% of CCR5 alleles in a pool of primary human CD4+ T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4+ T cells had lower viral loads and higher CD4+ T-cell counts than mice engrafted with wild-type CD4+ T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4+ T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN–modified autologous CD4+ T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

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Figure 1: ZFN-mediated disruption of CCR5 and protection from HIV-1 infection in GHOST-CCR5 cells.
Figure 2: In vitro selection of CCR5-disrupted cells following HIV-1 challenge of the CD4+ T-cell line, PM1.
Figure 3: Enrichment of CCR5 ZFN–modified primary CD4+ T cells during in vitro HIV-1 challenge.
Figure 4: 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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Acknowledgements

Research supported in part by National Institutes of Health, a grant from ATP NIST and the Abramson Family Cancer Research Institute. Elena Perez was supported by K08AI062468 for this work. The authors are grateful for constructive comments from Frederick Bushman, for help by Anthony Secreto and other lab members, for support from the Center for AIDS Research Cores, for advice from Bruce Levine and Gwen Binder, for bioinformatics support from Beilin Zhang, for analysis of the V3 loop data by Toby Dylan Hocking, and for experimental assistance from Gwenn-aël H. Danet-Desnoyers and the Xenograft Core Facility at the University of Pennsylvania School of Medicine, Erica Moehle, Jeremy Rock, Lei Zhang, Shuyuan Yao, Nhu Tran, Matthew Mendel, Deng Xia and Sarah Hinkley and members of the Sangamo production group, Melody Hung-Fan and the Contra Costa Public Health Lab for HIV RNA analyses, for pAdEasy-1/F35 vector provided by Xiaolong Fong, and at Bioqual Inc., Mark Lewis and Jake Yalley-Ogunro. CXCR4 tropic HIV-1BK132 and CCR5 tropic strains, US-1 were from John Mascola (Vaccine Research Center, NIH, Bethesda, Maryland), and Bal-1 was from Suzanne Gartner (Johns Hopkins, Baltimore). The following reagent was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: (GHOST (3) Hi-5 and GHOST (3) CXCR4) from Vineet N. KewalRamani and Dan R. Littman.

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  1. Yann Jouvenot

    Present address: Present address: Process Science Department, Bayer Hematology/Cardiology, 800 Dwight Way, Berkeley, California 94701, USA.,

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, 421 Curie Blvd., Room 554, BRB II/III, Philadelphia, Pennsylvania 19104-6160, USA.,

    Elena E Perez, Olga Liu, Carmine Carpenito, Richard G Carroll, James L Riley & Carl H June

  2. Division of Allergy and Immunology, Children's Hospital of Philadelphia, Joseph Stokes, Jr. Research Institute, 3615 Civic Center Blvd., Philadelphia, Pennsylvania 19104-4318, USA.,

    Elena E Perez & Jordan S Orange

  3. Sangamo BioSciences, Inc., Point Richmond Tech Center II, 501 Canal Blvd., Suite A100, Richmond, California 94804, USA.,

    Jianbin Wang, Jeffrey C Miller, Yann Jouvenot, Kenneth A Kim, Nathaniel Wang, Gary Lee, Victor V Bartsevich, Ya-Li Lee, Dmitry Y Guschin, Igor Rupniewski, Adam J Waite, Fyodor D Urnov, Edward J Rebar, Dale Ando, Philip D Gregory & Michael C Holmes

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Contributions

E.E.P., Y.J., J.W., O.L., C.C., K.A.K., J.S.O., J.C.M., V.V.B., D.Y.G., I.R., A.J.W., Y.-L.L., N.W., G.L., F.D.U. and E.J.R. designed and performed experiments; R.G.C., D.A. and P.D.G. assisted with experimental design; J.L.R., M.C.H., P.D.G. and C.H.J. are co-senior authors; E.E.P., M.C.H., P.D.G. and C.H.J. wrote the manuscript.

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Correspondence to Carl H June.

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J.W., Y.J., J.C.M., K.A.K. N.W., G.L., V.V.B., Y.-L.L., D.Y.G., I.R., A.J.W., F.D.U., E.J.R., D.A., P.D.G. and M.C.H. are or were employees of Sangamo Biosciences.

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Perez, E., Wang, J., Miller, J. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat Biotechnol 26, 808–816 (2008). https://doi.org/10.1038/nbt1410

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