The purpose of this research program is to develop an approach for the treatment of AIDS by endowing the patient's own cells with stable resistance to HIV. This highly innovative technology involves the disruption of a gene that is required for HIV entry into cells, the CCR chemokine receptor, within the genome of the patient's hematopoietic stem cells (HSCs). Such ablation is achieved by invoking a DNA repair pathway known as end joining, to heal a double-strand break introduced in vivo at the CCR5 locus by an enzyme engineered for that purpose, or a zinc finger protein endonuclease (ZFN). End joining is an extensively studied phenomenon that can result in loss-of function alleles. Relevant to this research program, humans with a naturally occurring loss-of-function deletion in both CCR5 alleles are resistant to infection with M-tropic strains of HIV-1. Therapeutic application of the ZFN-based technology will focus on interrupting the CCR5 gene locus in HSCs isolated from the bone marrow of infected patients, and then infusing the HIV-1 resistant cells into the original donors. We have built ZFNs that disrupt the endogenous CCR gene on human cells with very high efficiency. The proposed research program has the following goals. First, we will use these ZFNs to disrupt the CCR5 gene in human HSCs using the non-integrating lentiviral delivery systems we have developed. Second, we will study the maintenance of pluripotency by the HSCs following the gene disruption procedure and will measure the frequency of the CCR5-negative progeny generated from these cells. Lastly, we will extensively study the consequences of ZFN-mediated disruption on genome integrity and function. Successful completion of these goals could pave the way for more comprehensive safety and efficacy studies in the context of preclinical developmental efforts. Because the global impact of HIV-1 infection/AIDS is enormous, there is a major need to develop new antiretroviral therapeutic approaches. This proposal intends to develop a revolutionary way to treat, and potentially, cure AIDS, by using gene disruption to confer HIV-1 resistance to the patients' HSCs. The treatment procedure we plan to develop would involve taking cells from the patient's immune system, changing their DNA to make it HIV-1 resistant, and then introducing them into the patient, where they would combat the virus while remaining uninfected
