Gene editing using a zinc-finger nuclease mimicking the CCR5Δ32 mutation induces resistance to CCR5-using HIV-1

To characterize a new zinc-finger nuclease (ZFN) that targets close to the sequence of the 32 bp deletion polymorphism in the CCR5 gene, and to generate cells resistant to HIV-1 strains that use CCR5. CCR5 Delta 32 is a naturally occurring deletion that provides genetic resistance to R5-tropic HIV-1. The specificity and efficacy of a newly identified target for CCR5 gene editing, near the CCR5 Delta 32 sequence (ZFNCCR5 Delta 32), was assessed as well as its ability to generate cells resistant to HIV infection with reduced off-target effects. ZFNCCR5 Delta 32 activity was evaluated by heteroduplex formation in human K562 cells. Assessment of ZFNCCR5 Delta 32 specificity was analysed in silico. The yield of ZFNCCR5 Delta 32 in cell culture was improved by fluorescence-activated cell sorting, and the anti-HIV potency of ZFNCCR5 Delta 32 was measured in vitro in TZM-bl cells against HIV-1 strains. ZFNCCR5 Delta 32 effectively recognized the CCR5 Delta 32 region, inducing a frameshift of the CCR5 coding region that resulted in the complete absence of CCR5 expression of mRNA and of protein at the cell surface. CCR5 knockout cells were refractory to HIV-1 infection by the R5-using strain BaL. Unlike previous CCR5 ZFN studies, the new ZFN has no detectable off-target activity. ZFNCCR5 Delta 32 is a specific and efficient tool for the generation of CCR5 knockouts. Its ability to mimic the natural CCR5 Delta 32 phenotype in the absence of relevant off-site cutting events suggests that ZFNCCR5 Delta 32 might be safe in clinical research.