1740-P: Gene-Editing of FOXP3 in Antigen-Specific CD4⁺ T Cells for Restoration of Immune Tolerance in Type 1 Diabetes

Clinical trials for type 1 diabetes (T1D) using expanded, autologous natural regulatory T cells (nT_reg) have demonstrated safety and sustained engraftment. Limitations to this approach include the requirement for potential instability in vivo, lack of antigen specificity, and intrinsic T_reg defects in some patients. As an alternative strategy, we used gene editing to convert primary human or mouse CD4⁺T cells into immunosuppressive T_reg-like cells. Our approach utilizes co-delivery of designer nucleases and AAV6 homology-directed repair templates to enforce the expression of FOXP3, a transcription factor determining T_reg development. These edited (ed)T_reg exhibit a phenotype and functional activity similar to nT_reg and ameliorate the inflammatory xenograft response of effector T cells in NOD-scid-Il2rg^null (NSG) mice. In the current study, we hypothesized that antigen-specific edT_reg would exhibit increased potency in treatment of autoimmune diseases including T1D. As an initial test of feasibility, we generated edTreg from flu- or tetanus-specific human CD4⁺ T cells and demonstrated antigen-specific suppressive activity. Next, we show proof-of-concept for antigen (Ag)-specific edT_reg in a T1D mouse model. Using mouse-specific nucleases and donor templates, we edited CD4⁺T cells from transgenic NOD mice expressing the diabetogenic, BDC2.5, TCR. BDC2.5 edT_reg were similar to nT_reg on the basis of key cell markers and cytokine production. BDC2.5 edT_reg or polyclonal edT_reg from NOD mice were co-transferred with effector BDC2.5 T cells (CD4⁺CD25^-) into NSG mice. Recipients of only effector cells exhibited rapid diabetes onset. In contrast, edTreg delayed disease and, as hypothesized, Ag-specific edT_reg exhibited much greater suppression and longer delays in T1D onset. Together, our findings demonstrate the capacity to generate functional, Ag-specific, edT_reg with potential therapeutic application in promoting immune tolerance in T1D.