CCR5 is the major co-receptor for HIV-1 entry and an important target for drug development. The recent finding that hematopoietic stem cell [1] transplantation from a CCR5-negative donor to an HIV-infected patient produces long-term virus control in the absence of antiretroviral drugs suggests the potential of stem cell and gene therapies targeting CCR5. To this end, we are developing a protocol to knock-out CCR5 in a patient's own HSC using CCR5-targeted zinc finger nucleases (ZFNs).

ZFNs are sequence specific proteins that generate a double-stranded break in DNA, which is converted into a gene-disrupting lesion by host repair processes. We have optimized the delivery and function of ZFNs targeting CCR5 in human CD34-positive HSC, achieving up to 27% disruption of CCR5 alleles. We confirmed that ZFN-treated HSC remain fully functional by transplanting a mouse model of human hematopoiesis, the NOD/SCID/IL2γcnull (NSG) mouse, where the modified HSC retained the ability to differentiate into multiple hematopoietic lineages.

Humanized NSG mice are additionally capable of supporting HIV-1 infection. Following challenge with an R5-tropic virus, control animals demonstrated altered CD4:CD8 ratios, profound loss of human cells in the thymus and GALT, and high viral loads in multiple tissues sampled. In contrast, ZFN-treated animals had significantly lower acute viral loads and very low levels of virus in tissues by 10-12 weeks post-infection. At this stage the numbers of human cells in tissues that are targets for HIV-1 infection had normalized, including the GALT and thymus. FACS and PCR analysis revealed a rapid and dramatic selection for CCR5-negative cells in these populations. These findings demonstrate that ZFN-treated HSC can generate HIV-resistant cells in vivo that rapidly replace cells depleted by HIV-1 infection, and importantly, preserve GALT populations. Transient ZFN treatment resulting in permanent disruption of CCR5 in autologous HSC could therefore represent a viable clinical approach for HIV-infected patients.