Major histocompatibility complex gene editing to prevent hematopoietic stem cell graft rejection - PROJECT SUMMARY/ABSTRACT Hematopoietic stem cell transplantation (HSCT) cures a large number of disorders caused by dysfunction of blood-based cells. However, only the most severe cases undergo transplant due to risks of treatment-related mortality (TRM). Graft-versus-host disease (GVHD), chemotherapy conditioning toxicity, and infections due to low white blood cell counts from myeloablative conditioning are the primary drivers of TRM. Depleting T cells from stem cell grafts markedly reduces GVHD, and conditioning the patient with anti-CD117 antibodies instead of chemotherapy reduces organ toxicity and preserves the infection-fighting abilities of white blood cells. Thus, the majority of HSCT TRM for could be resolved with a treatment paradigm combining T cell depletion from stem cell grafts with anti-CD117 conditioning. However, preservation of host immune cells with this paradigm will lead to rejection of donor stem cells. This project will answer critical questions related to new strategies for preventing immune rejection of the stem cell graft. Specifically, we seek to investigate human leukocyte antigen (HLA) matching between donor and recipient and develop ways to circumvent graft rejection due to mismatches. The overarching hypothesis we will test in this proposal is that silencing of mismatched HLA by gene editing will prevent immune-mediated rejection and enable stable engraftment of donor stem cells. This will be explored in the context of HSCT with haploidentical related donors. Haploidentical related donors are HLA matched with the patient at a minimum of half of the major HLA alleles, and are a near universally available donor type that addresses the issue of most non-Caucasian patients not having full HLA match options in the National Marrow Donor Program. In order to advance our long-term goal of developing HLA gene editing for prevention of graft rejection in haploidentical related donor HSCT, we have determined that our efforts will need to be guided by first answering 3 biological questions. Our first aim will be to test whether the reduction in DNA double strand break signaling of next generation gene editing methods will yield greater HSC fitness and engraftment potential compared to conventional Cas9-mediated knockout methods. Our second aim will be to determine whether reductions in cell surface HLA through gene editing will affect antigen presentation to a degree where T cell responses are negatively impacted. Our third aim will use a novel mouse model that recapitulates the genetics of related donor haploidentical transplant to determine if silencing of mismatched major histocompatibility complex is sufficient for preventing graft rejection, or if additional interventions to address NK cell, myeloid, and minor histocompatibility complex contributions to graft rejection are needed. Upon completion of these aims, our results will reveal critical information guiding the development of next generation HSCT approaches aimed at reducing TRM and thereby expanding the number of patients and disease indications eligible for curative HSCT.
