title: Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.
abstract: HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
authors.forename: Craig B
authors.initials: CB
authors.lastname: Wilen
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: John C
authors.initials: JC
authors.lastname: Tilton
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Kim
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Scott A
authors.initials: SA
authors.lastname: Sherrill-Mix
authors.forename: Sean C
authors.initials: SC
authors.lastname: Patro
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Secreto
authors.forename: Andrea P O
authors.initials: AP
authors.lastname: Jordan
authors.forename: Gary
authors.initials: G
authors.lastname: Lee
authors.forename: Joshua
authors.initials: J
authors.lastname: Kahn
authors.forename: Pyone P
authors.initials: PP
authors.lastname: Aye
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Bunnell
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Lackner
authors.forename: James A
authors.initials: JA
authors.lastname: Hoxie
authors.forename: Gwenn A
authors.initials: GA
authors.lastname: Danet-Desnoyers
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
authors.forename: James L
authors.initials: JL
authors.lastname: Riley
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Carl H
authors.initials: CH
authors.lastname: June
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Robert W
authors.initials: RW
authors.lastname: Doms
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism / Q000637:transplantation; D049109:Cell Proliferation; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004195:Disease Models, Animal; D005818:Genetic Engineering; D015658:HIV Infections / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000628:therapy; D015497:HIV-1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D019713:Receptors, CCR5 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014182:Transplantation, Autologous; D014183:Transplantation, Heterologous; D053586:Virus Internalization
chemical_list: C527276:CXCR4 protein, human; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D003851:Deoxyribonucleases