Background:  HIV requires the CD4 co-receptors CCR5 or CXCR4 to infect its target cells. Loss of HIV co-receptors is therefore an attractive therapy for HIV patients, yet no current methods permit the efficient therapeutic disruption of a chosen gene in the human genome. We have developed designed zinc-finger protein nucleases (ZFN) to target the CCR5 gene and create a double-strand break (DSB) at predetermined sequences. Natural DNA repair pathways, including non-homologous end joining, subsequently repair the DSB. This repair is error prone and thus results in permanent disruption of the target gene.

Methods:  Designed zinc-finger proteins that recognize coding sequences within the CCR5 gene were fused to the FokI catalytic domain to create ZFN in which the DNA binding specificity of the ZFP determines DSB location. ZFN binding and cleavage was determined in vitro using ELISA-based DNA binding assays and PAGE. Vectors encoding the ZFN were introduced into human cells for in vivo assessment of function. TGD was measured by determining the sequence at the targeted site.

Results:  Cell-based assays revealed that ZFN generated DSB in vivo leading to high-efficiency target gene disruption in transiently transfected cells even in the absence of selective pressure. These results have been confirmed in relevant primary cell types (monocytes, CD4⁺ T cells, CD34⁺ hematopoietic precursors). We also demonstrated that a cell line modified by CCR5-targeting ZFN became resistant to HIV infection. Challenge assays using T cells pre-treated with the ZFN are in progress, although it is well established that a patient’s cells carrying a deletion within the CCR5 gene are resistant to infection despite repeated exposure to the R5-tropic virus.

Conclusions:  The frequency of gene disruption observed supports its examination as a possible method for the therapeutic modification of isolated patient cells to generate HIV-resistant T cells or hematopoietic precursors.