Experimental Protocol and Results
Engineering HIV-Resistant Cells by CXCR4 Inactivation
In Wilen et al.'s study, untouched CD4+ T cells from normal donors are isolated from fresh blood using the RosetteSep™ Human CD4+ T Cell Enrichment Cocktail. Wilen et al. then transduce these cells with a viral vector for CXCR4-inactivating ZFNs (X4-ZFNs). In order to test whether this transduction conferred HIV-1 resistance, Wilen et al. challenge the CD4+ T cells in vitro with strains of HIV-1 known to utilize either CCR5 or CXCR4. As expected, transduced CD4+ T cells were resistant to HIV-1 strains known to use CXCR4, but not to those that used CCR5 to enter cells. Furthermore, CD4+ T cells that were homozygous for the CCR5-inactivating ccr5Δ32 allele and transduced with X4-ZFNs were resistant to both types of HIV-1. This suggests that inactivating CCR5 and CXCR4 simultaneously may be an effective treatment for HIV infection.
A Humanized Mouse Model for HIV-Resistant CD4+ T Cells
To investigate whether X4-ZFNs conferred HIV-1 resistance in vivo as well as in vitro, Wilen et al. injected a group of mice with X4-ZFN-treated human CD4+ T cells isolated using RosetteSep™, and a separate group of mice with R5-ZFN-treated human CD4+ T cells. 28 days later, they injected all mice with autologous CD4+ T cells previously infected with X4 HIV (a strain of HIV that uses CXCR4 to enter cells).
After 34 days, Wilen et al. isolated CD4+ T cells from mouse peripheral blood and spleens by positive selection using RoboSep™. They then compared the frequency of CXCR4 disruption in HIV-infected mice compared to control mice. CXCR4 disruption was significantly greater in HIV-infected mice, which suggests that HIV infection caused selection in favor of X4-ZFN-transduced (i.e. X4 HIV-resistant) cells (Figure 4).
By monitoring human CD4+ T cell counts in both groups of mice, Wilen et al. found that treatment with X4-ZFN conferred significant protection to HIV, but that this protection waned over time. Wilen et al. sequenced viral RNA isolated from mouse plasma after the experiment, and identified a mutation in viral Env isolated from X4-ZFN mice, which likely enabled HIV to use CCR5. This suggests that the virus rapidly evolved in vivo to use CC5R in place of CXCR4, which explains the transient nature of HIV resistance in their model.