Manufacture and In Vivo Support of Gene-Edited, Regulatory-like, T-Cells (edTreg) Using a Synthetic IL2 Receptor

Regulatory T-cells (Treg) depend on signals from IL2 and their endogenous T-cell receptors to survive, proliferate, and maintain suppressive activity. We have developed a strategy for engineering effector CD4 T-cells into edited, regulatory-like T-cells (edTregs) for treatment of autoimmune disease. edTregs contain a rapamycin-activated synthetic IL2 receptor (designated a chemical-induced signaling complex or CISC) for use in selective cell manufacturing and in vivo trophic support. Our strategy uses gene editing to integrate an MND/ CISC expression cassette into the native FOXP3 locus, resulting in the CISC system cis-linked to expression of the native FOXP3 gene. The resulting high-level and stable expression of FOXP3 converts peripheral T-cells to edTregs with immunosuppressive activity. Cis-linked expression of the CISC system allows for selective cell expansion and in vivo support using rapamycin. Using an optimized protocol, we obtained efficient HDR rates across cells from multiple healthy donors. Edited cells were enriched to >90% purity and expanded 20-50-fold over a 10 day period of culture in rapamycin. Importantly, we demonstrated sustained in vivo suppressive activity of edTregs in a xeno-GvHD mouse model, and successful trophic support of these edTregs by rapamycin (via the CISC) in the absence of effector CD4 T-cells. Along with preliminary data showing successful editing of CD4-positive T-cells from auto-immune disease patients, our data provide pre-clinical proof-of-concept data supporting clinical use of CISC-edTreg in conjunction with rapamycin support for therapy of autoimmune disease.