Clinical trials for type 1 diabetes (T1D) using expanded, autologous natural regulatory T cells (nTreg) have demonstrated safety and sustained engraftment. Limitations to this approach include the requirement for potential instability in vivo, lack of antigen specificity, and intrinsic Treg defects in some patients. As an alternative strategy, we used gene editing to convert primary human or mouse CD4+T cells into immunosuppressive Treg-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 Treg development. These edited (ed)Treg exhibit a phenotype and functional activity similar to nTreg and ameliorate the inflammatory xenograft response of effector T cells in NOD-scid-Il2rgnull (NSG) mice. In the current study, we hypothesized that antigen-specific edTreg 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 edTreg 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 edTreg were similar to nTreg on the basis of key cell markers and cytokine production. BDC2.5 edTreg or polyclonal edTreg 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 edTreg exhibited much greater suppression and longer delays in T1D onset. Together, our findings demonstrate the capacity to generate functional, Ag-specific, edTreg with potential therapeutic application in promoting immune tolerance in T1D.
Y. Xiang: None. K. Sommer: None. Y. Honaker: None. J. Smith: None. T.R. Torgerson: Consultant; Self; CSL Behring, Grifols, Shire, UCB, Inc. Research Support; Self; CSL Behring. J. Buckner: None. D. Rawlings: None.
Seattle Children’s Research Institute, Seattle Children's Center for Immunity and Immunotherapies; Seattle Children's Program in Cell and Gene Therapy; Seattle Children's Guild Association (to D.R.); The Leona M. and Harry B. Helmsley Charitable Trust (to D.R.); National Institutes of Health (DP3DK111802-01 to D.R.)