Generation of islet antigen -specific engineered Tregs for use in rlD therapy via homology-directed gene editing of conventional CD4+T cells

Abstract Adoptive transfer of regulatory T cells (Tregs) is therapeutic in T1D mouse models. Notably, Tregs specific for pancreatic islets were shown more potent than polyclonal Tregs in blocking disease. However, the frequency of antigen-specific Tregs is extremely low and ex vivo expansion has the potential risk to destabilize Tregs leading to an effector phenotype. Here, we developed methods to generate durable, antigen-specific engineered Tregs (EngTregs) from primary human CD4+ T cells using a combination of lentiviral TCR transduction and FOXP3 homology-directed repair (HDR)-editing. Using TCRs derived from clonally expanded islet-reactive CD4+ T cells in T1D, we generated islet-specific EngTregs that exhibit a Treg-like phenotype. Islet-specific EngTregs effectively suppress proliferation and cytokine production by islet-specific effector T cells (Teff). Notably, EngTregs suppress Teff recognizing the identical peptide as well as bystander Teff recognizing alternative islet antigens. Importantly, islet-specific EngTregs suppress polyclonal endogenous islet-specific T cells derived from PBMC of individuals with T1D. To directly assess the capacity of EngTregs to modulate diabetes in vivo, we established an identical HDR-editing strategy in islet-specific murine cells. Following adoptive transfer, islet-specific mu-EngTregs homed to the pancreas and blocked diabetes triggered by islet-specific Teff in recipient mice. Collectively, our approach enables the production of EngTregs specific to pancreatic islets with the capacity to efficiently suppress islet specific responses. This approach has the capacity to deliver targeted islet specific therapy to treat or prevent T1D.