Figure 6. Treatment with X4-ZFNs confers partial protection to HIV-1 in humanized mice in vivo.

NSG mice were injected with human CD4+ T cells treated with X4-ZFNs or R5-ZFNs. 28 days post injection, mice were infected with primary X4 HIV-1 (Bk132) or were mock-infected. (A) CD4+ T cell counts were measured every 7–10 days post infection. In the presence of Bk132, treatment with X4-ZFNs conferred protection at 14 d.p.i (p = .05); however, this protection wanes by 34 d.p.i. (p = .88) (B) Cxcr4 disruption frequency was assessed by the surveyor nuclease assay in both peripheral blood (p<.001) and spleen (p<.001). At day 34 post infection, human CD4+ T cells were purified by positive selection prior to analysis to reduce any bias from low frequency contaminating human cells. Only samples with a detectable PCR signal are shown. Disruption frequency did not deviate significantly from the cell innoculum in either the blood or spleen. Data in (A) and (B) were analyzed by a general estimating equation (GEE). (C) HIV-1 Env from X4-ZFN mouse plasma was sequenced revealing a consensus Y302N mutation. To evaluate coreceptor tropism, a representative Env from the X4-ZFN mice and the viral innoculum were pseudotyped and used to infect NP2 cell lines expressing CD4 and either CCR5 or CXCR4. R5 HIV-1 (JRFL), R5X4 HIV-1 (R3A), and X4 HIV-1 (TYBE) controls are shown. Infectivity on NP2/CD4/CXCR4 cells was divided by that on NP2/CD4/CCR5 cells to determine relative coreceptor use. Data is an average of three independent experiments each done in triplicate. Error bars represent standard error.