Project Summary/Abstract Over the last decade, pancreatic islet transplantation has become a consistent and effective minimally invasive approach to restore normoglycemia in patients with type 1 diabetes1. Despite recent advances, widespread application has been limited by several barriers, including (1) poor survival and engraftment of islets following infusion into the portal vein of the liver, secondary to instant blood-mediated inflammatory reaction (IBMIR) and ischemia, (2) an inability to consistently achieve long-term insulin independence despite multiple infusions of islets from up to three donors, (3) a critical shortage of donor islets available for transplant, and (4) the inability to retrieve the graft in real time in case of adverse events. This proposal aims to address all four of these barriers utilizing genome-engineered stem-cell-derived beta cells. In contrast to cadaveric islets, human pluripotent stem cell-derived beta (SC-beta) cells represent a replenishable source of replacement beta cells2–7. Improving engraftment of SC-beta cells in an extra-hepatic site, together with engineering safety switches to delete any implanted SC-derived cells that display aberrant growth, will render this therapy safer and more effective, bringing benefit to more patients. Our prior work has shown that co-transplantation of parathyroid gland tissue with adult donor islets improves survival and engraftment at a retrievable intramuscular injection site in mice. We hypothesize that the secreted factors uniquely expressed by parathyroid gland may improve islet survival and vascularization. However, to overcome the limitations of parathyroid gland donor tissue availability and procurement, these effective factors will be introduced into stem-cell-derived beta cells using genome engineering strategies to improve engraftment and angiogenesis. Moreover, because the potential for outgrowth or oncogenesis is a major safety concern with any stem-cell-derived therapy, we will dually incorporate inducible safety switches that will allow for small molecule-driven clearance of residual pluripotent cells following beta cell differentiation, as well as clearance of the entire graft in the instance of an adverse event. These approaches will be vetted by transplanting engineered stem-cell-derived beta cells into immunodeficient mice that have induced diabetes, to directly assess the disease-modifying activity of these implanted cells in a physiological context. Ultimately, this work will advance the therapeutic potential of beta cell transplantation and address the major current clinical bottlenecks that prevent stem-cell-derived beta cell therapy from becoming a universal treatment strategy for diabetes.