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Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo

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Abstract

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34+ hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rγnull mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4+ T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5−/− cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5−/− HSPCs can populate an infected animal with HIV-1-resistant, CCR5−/− progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

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Figure 1: ZFN-mediated disruption of CCR5 in CD34+ HSPCs.
Figure 2: Protection of human CD4+ T cells in peripheral blood of HIV-infected mice previously engrafted with ZFN-modified CD34+ HSPCs.
Figure 3: Effects of HIV-1 infection on human cells in HSPC-engrafted NSG mice.
Figure 4: HIV-1 infection selects for disrupted CCR5 alleles.
Figure 5: ZFN activity produces heterogeneous mutations in CCR5.
Figure 6: Control of HIV-1 replication in mice receiving ZFN-treated CD34+ HSPCs.

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Change history

  • 20 July 2010

    In the version of this article initially published online, the callout to Figure 6b was written incorrectly as Figure 6c. Also, in Figure 2b, the label on the y axis was missing a “/” between CD4+ and CD8+. The errors have been corrected for the print, PDF and HTML versions of this article.

References

  1. Wu, L. et al. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature 384, 179–183 (1996).

    CAS  PubMed  Google Scholar 

  2. deRoda Husman, A.M., Blaak, H., Brouwer, M. & Schuitemaker, H. CC chemokine receptor 5 cell-surface expression in relation to CC chemokine receptor 5 genotype and the clinical course of HIV-1 infection. J. Immunol. 163, 84597–84603 (1999).

    Google Scholar 

  3. Samson, M. et al. Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382, 722–725 (1996).

    CAS  PubMed  Google Scholar 

  4. Novembre, J. et al. The geographic spread of the CCR5 Delta32 HIV-resistance allele. PLoS Biol. 3, e339 (2005).

    PubMed  PubMed Central  Google Scholar 

  5. Glass, W.G. et al. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J. Exp. Med. 203, 35–40 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Kantarci, O.H. et al. CCR5Δ32 polymorphism effects on CCR5 expression, patterns of immunopathology and disease course in multiple sclerosis. J. Neuroimmunol. 169, 137–143 (2005).

    CAS  PubMed  Google Scholar 

  7. Rossol, M. et al. Negative association of the chemokine receptor CCR5 d32 polymorphism with systemic inflammatory response, extra-articular symptoms and joint erosion in rheumatoid arthritis. Arthritis Res. Ther. 11, R91–98 (2009).

    PubMed  PubMed Central  Google Scholar 

  8. Dau, B. & Holodiny, M. Novel targets for antiretroviral therapy: clinical progress to date. Drugs 69, 31–50 (2009).

    CAS  PubMed  Google Scholar 

  9. Hutter, G. et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N. Engl. J. Med. 360, 692–698 (2009).

    PubMed  Google Scholar 

  10. Hutter, G., Schneider, T. & Thiel, E. Transplantation of selected or transgenic blood stem cells—a future treatment for HIV/AIDS? J. Int. AIDS Soc. 12, 10–14 (2009).

    PubMed  PubMed Central  Google Scholar 

  11. Anderson, J. et al. Safety and efficacy of a lentiviral vector containing three anti-HIV genes–CCR5 ribozyme, tat-rev siRNA, and TAR decoy–in SCID-hu mouse-derived T cells. Mol. Ther. 15, 1182–1188 (2007).

    CAS  PubMed  Google Scholar 

  12. Bai, J. et al. Characterization of anti-CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo. Mol. Ther. 1, 244–254 (2000).

    CAS  PubMed  Google Scholar 

  13. Kumar, P. et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134, 577–586 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Swan, C.H. et al. T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery. Gene Ther. 13, 1480–1492 (2006).

    CAS  PubMed  Google Scholar 

  15. Swan, C.H. & Torbett, B.E. Can gene delivery close the door to HIV-1 entry after escape? J. Med. Primatol. 35, 236–247 (2006).

    CAS  PubMed  Google Scholar 

  16. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  PubMed  Google Scholar 

  17. Jasin, M. et al. Genetic manipulation of genomes with rare-cutting endonucleases. Trends Genet. 12, 224–228 (1996).

    CAS  PubMed  Google Scholar 

  18. Sonoda, E. et al. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst.) 5, 1021–1029 (2006).

    CAS  Google Scholar 

  19. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Ishikawa, F. et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 106, 1565–1573 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).

    CAS  PubMed  Google Scholar 

  22. Hollis, R.P. et al. Stable gene transfer to human CD34(+) hematopoietic cells using the Sleeping Beauty transposon. Exp. Hematol. 34, 1333–1343 (2006).

    CAS  PubMed  Google Scholar 

  23. Sumiyoshi, T. et al. Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system. Hum. Gene Ther. 20, 1607–1626 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Mátés, L. et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 41, 753–761 (2009).

    PubMed  Google Scholar 

  25. Xue, X. et al. Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system. Blood 114, 1319–1330 (2009).

    CAS  PubMed  Google Scholar 

  26. Basu, S. & Broxmeyer, H.E. CCR5 ligands modulate CXCL12-induced chemotaxis, adhesion, and Akt phosphorylation of human cord blood CD34+ cells. J. Immunol. 183, 7478–7488 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Watanabe, S. et al. Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses. Blood 109, 212–218 (2007).

    CAS  PubMed  Google Scholar 

  28. Brenchley, J.M. et al. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J. Exp. Med. 200, 749–759 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Brenchley, J.M. et al. HIV disease: fallout from a mucosal catastrophe? Nat. Immunol. 7, 235–239 (2006).

    CAS  PubMed  Google Scholar 

  30. Guadalupe, M. et al. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J. Virol. 77, 11708–11717 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Talal, A.H. et al. Effect of HIV-1 infection on lymphocyte proliferation in gut-associated lymphoid tissue. J. Acquir. Immune Defic. Syndr. 26, 208–217 (2001).

    CAS  PubMed  Google Scholar 

  32. Li, Q. et al. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434, 1148–1152 (2005).

    CAS  PubMed  Google Scholar 

  33. Mattapallil, J.J. et al. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature 434, 1093–1097 (2005).

    CAS  PubMed  Google Scholar 

  34. Veazey, R.S. et al. Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science 280, 427–431 (1998).

    CAS  PubMed  Google Scholar 

  35. Berges, B.K. et al. HIV-1 infection and CD4 T cell depletion in the humanized Rag2−/−gamma c−/− (RAG-hu) mouse model. Retrovirology 3, 76–90 (2006).

    PubMed  PubMed Central  Google Scholar 

  36. Appay, V. & Sauce, D. Immune activation and inflammation in HIV-1 infection: causes and consequences. J. Pathol. 214, 231–241 (2008).

    CAS  PubMed  Google Scholar 

  37. Stoddart, C.A. et al. IFN-alpha-induced upregulation of CCR5 leads to expanded HIV tropism in vivo. PLoS Pathog. 6, e1000766 (2010).

    PubMed  PubMed Central  Google Scholar 

  38. Choudhary, S.K. et al. R5 human immunodeficiency virus type 1 infection of fetal thymic organ culture induces cytokine and CCR5 expression. J. Virol. 79, 458–471 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Kahn, J.O. & Walker, B.D. Acute human immunodeficiency virus type 1 infection. N. Engl. J. Med. 339, 33–39 (1998).

    CAS  PubMed  Google Scholar 

  40. Margolick, J.B. et al. Impact of inversion of the CD4/CD8 ratio on the natural history of HIV-1 infection. J. Acquir. Immune Defic. Syndr. 42, 620–626 (2007).

    Google Scholar 

  41. Henrard, D.R. et al. Natural History of HIV-1 cell-free viremia. J. Am. Med. Assoc. 274, 554–558 (1995).

    CAS  Google Scholar 

  42. Chen, R.Y. et al. Distribution of health care expenditures for HIV-infected patients. Clin. Infect. Dis. 42, 1003–1010 (2006).

    CAS  PubMed  Google Scholar 

  43. Richman, D.D. et al. The challenge of finding a cure for HIV infection. Science 323, 1304–1307 (2009).

    CAS  PubMed  Google Scholar 

  44. Rossi, J.J., June, C.H. & Kohn, D.B. Genetic therapies against HIV. Nat. Biotechnol. 25, 1444–1454 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Bibikova, M. et al. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161, 1169–1175 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Doyon, Y. et al. Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases. Nat. Biotechnol. 26, 702–708 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Santiago, Y. et al. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc. Natl. Acad. Sci. USA 105, 5809–5814 (2008).

    CAS  PubMed  Google Scholar 

  48. Peters, W., Dupuis, M. & Charo, I.F. A mechanism for the impaired IFN-gamma production in C–C chemokine receptor 2 (CCR2) knockout mice: Role of CCR2 in linking the innate and adaptive immune responses. J. Immunol. 165, 7072–7077 (2000).

    CAS  PubMed  Google Scholar 

  49. Smith, M.W. et al. CCR2 chemokine receptor and AIDS progression. Nat. Med. 3, 1052–1053 (1997).

    CAS  PubMed  Google Scholar 

  50. Davis, B.R. & Candotti, F. Revertant somatic mosaicism in the Wiskott-Aldrich syndrome. Immunol. Res. 44, 127–131 (2009).

    PubMed  Google Scholar 

  51. Hirschhorn, R. et al. Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency. Nat. Genet. 3, 290–295 (1996).

    Google Scholar 

  52. Hirschhorn, R. et al. In vivo reversion to normal of inherited mutations in humans. J. Med. Genet. 40, 721–728 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Stephan, V. et al. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N. Engl. J. Med. 335, 1563–1567 (1996).

    CAS  PubMed  Google Scholar 

  54. Chun, T.W. et al. Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. J. Infect. Dis. 197, 714–720 (2008).

    CAS  PubMed  Google Scholar 

  55. Lackner, A.A. et al. The gastrointestinal tract and AIDS pathogenesis. Gastroenterology 136, 1965–1978 (2009).

    PubMed  PubMed Central  Google Scholar 

  56. Picker, L.J. Immunopathogenesis of acute AIDS virus infection. Curr. Opin. Immunol. 18, 399–405 (2006).

    CAS  PubMed  Google Scholar 

  57. Veazey, R.S., Marx, P.A. & Lackner, A.A. The mucosal immune system: primary target for HIV infection and AIDS. Trends Immunol. 22, 626–633 (2001).

    CAS  PubMed  Google Scholar 

  58. Krishnan, A. et al. Autologous stem cell transplantation for HIV associated lymphoma. Blood 98, 3857–3859 (2001).

    CAS  PubMed  Google Scholar 

  59. Aiuti, A. et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–2413 (2002).

    CAS  PubMed  Google Scholar 

  60. Aiuti, A. et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N. Engl. J. Med. 360, 447–458 (2009).

    CAS  PubMed  Google Scholar 

  61. Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).

    CAS  PubMed  Google Scholar 

  62. Cartier, N. et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823 (2009).

    CAS  PubMed  Google Scholar 

  63. Biti, R. et al. HIV-1 infection in an individual homozygous for the CCR5 deletion allele. Nat. Med. 3, 252–253 (1997).

    CAS  PubMed  Google Scholar 

  64. Oh, D.Y. et al. CCR5Delta32 genotypes in a German HIV-1 seroconverter cohort and report of HIV-1 infection in a CCR5Delta32 homozygous individual. PLoS ONE 3, e2747–2753 (2008).

    PubMed  PubMed Central  Google Scholar 

  65. Weiser, B. et al. HIV-1 coreceptor usage and CXCR4-specific viral load predict clinical disease progression during combination antiretroviral therapy. AIDS 22, 469–479 (2008).

    PubMed  Google Scholar 

  66. Ogert, R.A. et al. Mapping Resistance to the CCR5 co-receptor antagonist vicriviroc using heterologous chimeric HIV-1 envelope genes reveals key determinants in the C2–V5 domain of gp120. Virology 373, 387–399 (2008).

    CAS  PubMed  Google Scholar 

  67. Soulie, C. et al. Primary genotypic resistance of HIV-1 to CCR5 antagonist treatment-naïve patients. AIDS 22, 2212–2214 (2008).

    CAS  PubMed  Google Scholar 

  68. Palmer, S. et al. Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy. Proc. Natl. Acad. Sci. USA 105, 3879–3884 (2008).

    CAS  PubMed  Google Scholar 

  69. Dinoso, J.B. et al. Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy. Proc. Natl. Acad. Sci. USA 106, 9403–9408 (2009).

    CAS  PubMed  Google Scholar 

  70. Mitsuyasu, R.T. et al. Phase 2 gene therapy trial of an anti-HIV ribozyme in autologous CD34+ cells. Nat. Med. 15, 285–292 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Shultz, L.D. et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hematopoietic stem cells. J. Immunol. 174, 6477–6489 (2005).

    CAS  PubMed  Google Scholar 

  72. Rouet, F. et al. Transfer and evaluation of an automated, low-cost real-time reverse transcription-PCR test for diagnosis and monitoring of human immunodeficiency virus type 1 infection in a West African resource-limited setting. J. Clin. Microbiol. 43, 2709–2717 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Sun, Z. et al. Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1. J. Exp. Med. 204, 705–714 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Loken, M.R. et al. Establishing lymphocyte gates for immunophenotyping by flow cytometry. Cytometry 11, 453–459 (1990).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank A. Cuddihy, S. Ge, R. Hollis and N. Smiley for expert technical assistance; C. Lutzko, V. Garcia, R. Akkina, B. Torbett and M. McCune for advice regarding humanized mice; and M. McCune for communicating unpublished data. This work was supported by funding from the California HIV/AIDS Research Project (P.M.C.), The Saban Research Institute (V.T.), and the National Heart, Lung, and Blood Institute P01 HL73104 (G.M.C., D.B.K. and P.M.C.).

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N.H. performed most of the experiments; J.W., K.K., G.F. and X.W. developed assays and analyzed samples; V.T. contributed to discussions; N.H., G.M.C., D.B.K., P.D.G., M.C.H. and P.M.C. designed the experiments and analyzed data; N.H. and P.M.C. wrote the manuscript.

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Correspondence to Paula M Cannon.

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J.W., K.K., G.F., P.D.G. and M.C.H. are employees of Sangamo BioSciences.

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Holt, N., Wang, J., Kim, K. et al. Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo. Nat Biotechnol 28, 839–847 (2010). https://doi.org/10.1038/nbt.1663

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