PROJECT SUMMARY / ABSTRACT Hematopoietic stem cell transplantation (HSCT) is potentially applicable to various medical settings beyond current standard of care (SOC), such as therapy for autoimmune disease, gene modification or induction of transplant tolerance. However, its wider clinical application is currently hampered by the myeloablative and genotoxic conditioning required for successful HSC engraftment. Such conditioning generally includes non- selective myeloablative treatments, such as total body irradiation (TBI), cyclophosphamide, or fludarabine that are associated with serious systemic side-effects including pancytopenia, infections, infertility, and, not infrequently, death. To develop a safer conditioning regimen without such myeloablative and genotoxic consequences, a therapeutic modality that selectively depletes host hematopoietic stem cells (HSCs) and opens a physical space in bone marrow (BM) niches needs to be identified. Based on murine studies, we hypothesized that selective inhibition of antiapoptotic B cell lymphoma 2 (Bcl-2) may effectively induce apoptosis of HSCs, thereby promoting HSC engraftment without need for non-selective myeloablative treatments. Indeed, a selective Bcl-2 inhibitor, Venetoclax, appeared significantly promotes hematopoietic chimerism by depleting HSCs in BM niches in non- human primates (NHPs). Although this was achieved without severe pancytopenia, some TBI was still required to induce chimerism as depletion of HSCs was limited with Venetoclax alone. Since Mcl-1, another anti-apoptotic protein, has recently been reported to be more critical for survival of HSCs, we further hypothesize that inhibition of Mcl-1 alone or in combination with Bcl-2 will more effectively deplete host HSCs, thereby creating sufficient space in BM niches and allowing optimal allogeneic HSC engraftment without any myeloablative treatment. The results of preliminary study have been encouraging, where successful induction of hematopoietic chimerism was achieved without TBI by combining low dose Mcl-1 inhibitor and Venetoclax. The main objective of this R21 project is to test whether Mcl-1 inhibition alone or with Bcl-2 co-inhibition can consistently and safely induce hematopoietic chimerism without TBI in MHC mismatched NHP HSCT. Chimerism induced by this approach will then be tested for induction of renal allograft tolerance. Since there is only limited information available on the role of different anti-apoptotic Bcl-2 proteins in primates, we will also characterize the intrinsic apoptotic pathways of HSCs in NHPs in this study.