38. CCR5 Gene Edited Hematopoietic Stem Cells Engraft in Diverse Anatomical Locales and Undergo SHIV-Dependent Positive Selection in Nonhuman Primates

Abstract

Background: Gene editing in hematopoietic stem/progenitor cells (HSPCs) has rapidly emerged as a promising therapy for a number of diseases, including human immunodeficiency virus (HIV) infection. We have previously demonstrated the feasibility of this approach in nonhuman primates. Here, we leverage our expertise with gene editing in large animal models to interrogate the clonal persistence, trafficking, and antiviral efficacy of CCR5-edited cells in the pigtailed macaque, M. nemestrina. Our objectives were to map the tissue distribution of HSPC-derived, gene edited progeny, understand how individual gene edited HSPCs persist following autologous transplantation, and develop strategies to select for these cells in vivo.Methods: Zinc Finger Nucleases (ZFNs) are used to target the CCR5 locus in macaque HSPCs. 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 simian/human immunodeficiency virus (SHIV). Gene edited HSPCs are transplanted either prior to SHIV infection, or in SHIV-infected animals that are treated with combination antiretroviral therapy (cART) in order to approximate a well-suppressed HIV+ patient. Edited cells are measured longitudinally in peripheral blood, bone marrow, gastrointestinal (GI) tract, and lymph nodes, and at necropsy in a panel of 25 tissues, using methods including deep sequencing.Results: We observe up to 14-fold enrichment of CCR5-gene edited memory CD4+ T-cells in SHIV-infected animals, consistent with virus-dependent selection against CCR5 wt memory CD4+ T-cells. Gene edited cells are found in a broad array of anatomical sites, including GI tract and lymph nodes. Spatial and temporal tracking of CCR5 mutations suggests that gene edited cells persist in an analogous fashion to control lentivirus gene-marked cells. Homology directed repair (HDR) pathways can be exploited in macaque CD34+ HSPCs, facilitating knock-in of selectable markers at the disrupted CCR5 locus.Conclusions: Our results in SHIV-infected animals reinforce that this gene editing strategy results in stable engraftment of CCR5-mutated and SHIV-resistant HSPCs and their progeny. Additionally. gene edited CD4+ T-cells undergo positive selection during active infection, further supporting the validity of this approach in the clinic. Moreover, our preliminary ex vivo HDR data suggest that these gene edited cells could be engineered to undergo virus-independent selection.