758. Towards Gene Knock out Therapy for AIDS/HIV: Targeted Disruption of CCR5 Using Engineered Zinc Finger Protein Nucleases (ZFNs).

HIV entry into target cells requires binding of the envelope protein GP120 to the CD4 co-receptors CCR5 or CXCR4. Loss of these HIV co-receptors is therefore attractive as a potential therapy for HIV patients, yet no current methods permit the efficient therapeutic disruption of a chosen gene in the human genome. To this end, we have developed designed zinc-finger protein nucleases (ZFNs) to target predetermined sequences within the CCR5 gene. Once these ZFNs create a double strand break (DSB) in the targeted DNA, natural DNA repair pathways, including Non-Homologous End Joining (NHEJ), subsequently repair the DSB. Importantly, however, NHEJ is error prone and thus can result in permanent disruption of the targeted gene.Designed ZFNs that recognize coding sequences within the CCR5 gene were generated and their DNA-binding and cleavage properties were determined in vitro. Vectors encoding the validated ZFNs were introduced into human cells for in vivo assessment of function. ZFNs targeted to the CCR5 co-receptor efficiently cleaved their expected DNA target sites, leading to high efficiency target gene disruption (>5%) 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 PM-1 and GHOST cells modified by the CCR5-ZFNs became resistant to HIV infection. Challenge assays using T cells pre-treated with these ZFNs are in progress, although it is well established that a patients cells carrying a deletion within the CCR5 gene are resistant to infection despite repeated exposure to R5-tropic virus. Moreover, 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.Molecular Therapy (2006) 13, S293–S293; doi: 10.1016/j.ymthe.2006.08.842 [ABSTRACT FROM AUTHOR]