Cells lacking both ccr5 and cxcr4 following R5/X4-ZFN treatment have a survival advantage in vivo in the presence of HIV. (A) The frequency of human ccr5- and cxcr4-modified genes significantly increases in the R5/X4-ZFN treatment group over the course of the in vivo infection. ccr5 and cxcr4 disruption are stable over time in the absence of HIV infection suggesting there is no significant adverse effect of dual ZFN treatment. We performed Illumina deep sequencing of the R5 and X4-ZFN target sites and identified ZFN-induced mutations at these sites in 8 of 9 uninfected and 8 of 9 infected animals. We were unable to obtain sequence information from 1 infected animal due to limiting quantities of CD4⁺ T cells, and killed 1 uninfected animal following the development of GVHD, and this animal was thus excluded. (B) We stained whole blood from infected and uninfected animals in the R5/X4-ZFN treatment group 34 days postinfection with human antibodies to identify human CD4⁺ T cells and determine surface levels of R5 and X4. Both CCR5 and CXCR4 expression were significantly reduced on dual ZFN-treated cells in the presence of HIV challenge suggesting that coreceptor negative cells have an in vivo survival advantage due to ZFN treatment.