Harnessing engineered T regulatory cells to promote beta cell health in T1D

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Summary Type 1 diabetes (T1D) is characterized by the destruction of β-cells, driven through autoimmune attack directed at the pancreatic islet. In concert with β-cell destruction, β-cell stress and injury contribute to disease by initiating enzymatic activities that compromise β-cell function and generate neoantigens. The investigators of this project will integrate knowledge of clinical T1D, β-cell stress, islet reactive T cells, regulatory T cells (Tregs) and gene engineering to create Tregs that will home to the islet and suppress autoimmunity and inflammation in a manner that will create an environment that will allow β-cell recovery and promote β-cell health. These engineered Tregs (EngTregs) will be generated utilizing homology-directed repair (HDR)-based gene editing to mediate constitutive expression of FOXP3 combined with lentiviral transduction of T cell receptor (TCR) sequences specific to antigens presented in the islet during periods of β-cell stress. Further, we propose develop EngTregs with the ability to co-deliver a payload (with or without TCR engagement) that will function to suppress ongoing inflammation and/or promote β-cell survival and growth. Aim 1 will generate stressed-islet-specific CD4+ and CD8+ EngTregs via gene editing and, in parallel, confer specificity to the pancreatic islet via expression of TCRs that recognize citrullinated or deamidated β-cell-derived neoepitopes. The central hypothesis for Aim 1 is that targeting neoantigens generated during periods of β-cell stress will enhance the targeted delivery of EngTregs to the site of tissue injury. Neoepitope discovery studies will identify TCR sequences restricted to novel β-cell stress neoepitopes. Functional studies will assess stressed-islet-specific EngTregs activation in the presence of β-cell stress as well as in the presence of CD4+ and CD8+ effector T cells. Aim 2 will generate islet-specific CD4+ and CD8+ EngTregs that mediate targeted immune suppression and co-deliver an islet-protective therapeutic cargo. In parallel, we will develop EngTregs that release their cargo upon TCR recognition of antigen. The proposed studies integrate primary human cell studies with relevant T1D murine models, facilitating more rapid identification of promising candidate engineered T cell products. The proposed studies are directly responsive to RFA-DK-21-005 with its request to 1) engineer antigen-specific Treg cells that can home to the pancreatic islet or pancreatic draining lymph nodes and inhibit effector T cells in these compartments; 2) engineer islet- homing synthetic suppressor cells, such as CD4+ T cells engineered to locally produce factors that dampen inflammation, inactivate effector T cells, or promote islet tissue repair; and 3) engineer T cells that produce factors with trophic effects on β-cells to promote function, immunoprotection and/or replication.