ZFNs simultaneously disrupt ccr5 and cxcr4 in primary human CD4⁺T cells. (A) Dual ZFN treatment does not result in a significant growth defect as determined by live primary CD4⁺ T-cell count following simultaneous delivery of increasing amounts of the R5- and X4-ZFNs. Data are from 1 of 3 independent experiments. (B) ccr5 and cxcr4 gene modification increases in a dose-dependent manner following R5/X4-ZFN treatment, as measured by the Cel1 assay 7 to 10 days post-ZFN treatment. Data are from 1 of 3 independent experiments. (C) Primary CD4⁺ T cells were sorted by CXCR4 expression into CXCR4-high and CXCR4-low populations (left) following simultaneous R5/X4-ZFN treatment. Successful sorting was confirmed by repeat FACS (right panel). Arrows indicate gating strategy. (D) ccr5 and cxcr4 gene disruption was measured by cel1 following R5/X4-ZFN treatment and subsequent sorting by FACS into CXCR4-high and -low populations. ccr5 disruption is similar in the X4-high and X4-low populations, suggesting ccr5 and cxcr4 disruption are not occurring in mutually exclusive cells. Data are from 1 of 3 independent experiments. (E) CD4⁺ T cells treated with equal MOIs of the AdGFP or the R5- and X4-ZFNs were stained with the memory markers CCR7 and CD45RO Tcm [CD45RO⁺ CCR7⁺] and Tem [CD45RO⁺CCR7⁻]). (F) ccr5 and cxcr4 gene modification in Tcm and Tem subsets was similar suggesting that long-lived Tcm cells can be efficiently rendered HIV-resistant. Data shown are from 1 of 3 independent experiments. Tcm, central memory T cell; Tem, effector memory T cell.