A. Specific Aims Type 1 diabetes (T1D) is a progressive autoimmune disease which renders individuals incapable of regulating their blood glucose levels due to immune-mediated β cell destruction, resulting in loss of insulin production and many severe health complications that, if untreated, lead to death. Careful monitoring of blood glucose coupled with insulin injections have made T1D a chronic disease in which T1D individuals live ~ a decade less than their healthy counterparts. Thus, a cure for T1D is highly desirable. Cell gene therapy has proven to be an effective way to treat recalcitrant diseases such as pediatric leukemia, where chimeric antigen receptor (CAR) expressing T cells achieve 90% complete response, putting many individuals into very long remissions1, but to date no engineered T cell therapies have been attempted to cure T1D in humans. We propose to test the hypothesis that engineered T cells can enable islet transplant with minimal or no additional immunosuppression. Islet transplantation represents the best-case scenario to test the ability of engineered T cells to protect islet cells from immune attack and will likely lay the foundation by which strategies are developed to treat new onset T1D. In islet transplant, MHC matching of donor and recipient rarely occurs, generating highly expressed, unique islet-specific HLA antigens that can be targeted by CAR engineered T regulatory cells (CAR Tregs) or T cells engineered to express molecules that suppress the immune system (T suppressor cells or Tsups). Additionally, through a comprehensive screening process, we have identified two targets, fibroblast activation protein (FAP) and dipeptidyl peptidase like 6 (DPP6), that are highly expressed on α and β cells and have limited expression elsewhere that could be used to treat recently diagnosed individuals, and all transplant recipients without the desired MHC mismatch. In this proposal, we will further develop this toolbox to both develop better in vivo, preclinical models of T1D and new cell and gene therapies that will prevent, stall or reverse T1D. Within the last decade, rapid progress made in T cell-based therapies makes it possible to consider such therapies for T1D. Following long-term remission of 3 cancer patients treated by CD19-specific CARs developed by the Center for Cellular Immunotherapies at Penn under the leadership of Carl June2, the CAR T cell revolution was launched. This early success drove considerable investment, empowering many institutions and companies to develop ways to improve both the safety and efficacy of, and reduce cost to produce engineered T cells. Many of these innovations will also help enable cell and gene therapies for T1D, which is the overarching goal of this RFA. The ultimate goal of this application is to successfully treat three non- human primates (NHP) with engineered T cells after an islet transplant, which we predict will launch similar enthusiasm for T1D cell and gene therapy as the f...