Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection.