ABSTRACT In this proposal we apply somatic cell gene editing strategies to enhance pancreatic beta cell replacement therapies for type 1 diabetes (T1D). We have formed a team that combines expertise in beta cell biology, synthetic and systems biology, and islet transplant immunology to address key impediments for efficient immunosuppression-free transplantation of pancreatic islets. We propose two orthogonal yet complementary aims to address two critical challenges in islet transplantation - islet survival and immune rejection. Most of the transplanted islets die before revascularization can occur, which limits the efficacy of the therapy. We have shown hypoxia and nutrient deprivation during ischemia independently and synergistically kill transplanted islet cells. Aim 1 of this proposal addresses the hypothesis that peri-transplant death can be alleviated by deleting negative regulators of beta cell survival or by over-expression of positive regulators. We will take both targeted and unbiased approaches to test candidate regulators and to identify novel regulators of human islet survival. Our team has already performed high-throughput screens using RNAi in primary human islets using in vivo transplant survival as a readout. We are ready to apply our expertise to CRISPRi and cDNA screens of primary human islets. Previous clinical islet transplant experiences show that stronger immunosuppression is associated with higher rate of insulin independence after islet transplantation. The immune system deploys multiple redundant mechanisms to eliminate transplanted foreign tissue. This, combined with the fragility of the transplanted islets and heightened immune functions in T1D recipients, forms a formidable immunological barrier to beta cell replacement therapy. We hypothesize that multipronged approach of minimizing islet cell immunogenicity, neutralizing inflammation in the graft, and blocking cellular infiltrate will shield the islets from immune rejection without the need for systemic immunosuppression. In Aim2, we will test this hypothesis by gene edit human islets to ablate the expression of polymorphic human leukocyte antigens. We will test dominant strategies that block innate inflammatory cytokines TNF, IL-1 and type 1 and type 2 interferons. We will also target adaptive immune cells by blocking their trafficking, activation and effector function. Successful confirmation of our hypotheses will provide proof-of-principle data to support efforts of clinical translation as next steps. We envision that these strategies may be applied to primary human islets, stem cell-derived beta cells, and even xenogeneic islets. While these CRISPR modalities are powerful research tools for screens and proof- of-concept experiments in the laboratory, base editing and/or prime editing may be preferred embodiments in the clinical setting. Our end goal is to generate game-changing strategies to address these key impediments, with a vision towards clinical translati...