684. Long-Term, Multilineage Engraftment of Zinc Finger Nuclease-Edited Hematopoietic Stem Cells in Nonhuman Primates

Abstract

Background: Nuclease-mediated gene editing in hematopoietic stem cells (HSCs) holds great promise for diseases including HIV infection and hemoglobinopathies, but little information is available regarding the feasibility of this approach in large animal models. To better evaluate the function of HSCs following gene editing, we have engineered cells with disrupted CCR5 alleles and assessed engraftment following autologous transplant in the pigtailed macaque, M. nemestrina. Disrupted CCR5 alleles in this model should directly protect against infection with simian/human immunodeficiency virus (SHIV). We are evaluating the extent to which CCR5-disrupted cell progeny engraft in macaques, and testing whether these cells impede infection by SHIV.Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSCs. Engraftment and persistence of these autologous stem cells, and stem cell-derived lymphoid and myeloid cells, are measured ex vivo and in vivo. Animals are challenged with SHIV virus containing an HIV envelope; to approximate the status of an HIV+ patient, three-drug combination antiretroviral therapy (cART) is initiated following viral set point. Animals reach undetectable levels of plasma viremia prior to autologous transplant with gene-edited cells.Results: CCR5 targeting experiments yield up to 60% gene disruption in CD34+ cells ex vivo, translating to approximately 5% steady state bulk disruption in vivo. Gene-disrupted cells demonstrate long-term, multilineage engraftment in macaques, including comparable levels of disruption in CD3+, CD20+, CD14+, and granulocyte subsets. We also observe biallelic disruption of CCR5 in colony forming assays. Importantly, this approach is equally feasible in SHIV-naive and in SHIV-infected, cART-suppressed animals. During robust SHIV replication, our preliminary data suggest that CCR5-deleted cells undergo positive selection in vivo.Conclusions: This is the first demonstration of successful long-term multilineage engraftment of ZFN-edited HSCs in a NHP transplantation model. The CCR5 gene editing/HSCT strategy presented here results in robust levels of target gene disruption in vivo, yet does not impair HSC engraftment or differentiation. CCR5-deleted cells can undergo positive selection following challenge with SHIV. Our model enables the evaluation of novel therapeutic approaches in a number of infectious and genetic diseases including HIV infection. Specifically, these results are applicable not only in the context of acute HIV exposure, but also in the clinically relevant setting of pre-existing latent HIV infection.