Primary CD4⁺T cells treated with the R5 and X4-ZFNs and challenged with HIV have a survival advantage in vitro. (A) Primary CD4⁺ T cells treated with GFP, the R5-ZFN, or 2 vectors encoding the R5 and X4-ZFNs were challenged with a mix of 2o HIV strains: the R5-using virus BaL and the X4-using virus HxB2. Dual ZFN treatment conferred a significant survival advantage in vitro compared with both the GFP and R5-ZFN control. Live cells were counted every 3 to 4 days for 32 days. Arrows indicate the time points where cells were reactivated with anti-CD3/CD28 magnetic beads. Data are from 1 of 3 independent experiments. (B) T7E1 analysis of ccr5 (top panel) and cxcr4 (bottom panel) gene modification reveals that HIV challenge enriches for ccr5 and cxcr4 disrupted cells. Analysis was performed 0 and 21 days postinfection. Data are from 1 of 3 independent experiments. (C) The proportion of modified ccr5 and cxcr4 alleles in cells treated with both ZFNs increases approximately twofold in the presence or absence of HIV, as measured by the T7E1 assay. Data are shown as the mean ± SEM of 3 independent experiments. P values calculated using the Student t test.