Gene editing to induce FOXP3 expression in human CD4+ T cells leads to a stable regulatory phenotype and function

Abstract

Thymic regulatory T cells (tT(regs)) are potent inhibitors of autoreactive immune responses, and loss of tT(reg) function results in fatal autoimmune disease. Defects in tT(reg) number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of T-reg as cell therapies to establish immune tolerance. Because tT(regs) are present at low numbers in circulating blood and may be challenging to purify and expand and also inherently defective in some subjects, we designed an alternative strategy to create autologous T-reg-like cells from bulk CD4(+) T cells. We used homology-directed repair (HDR)-based gene editing to enforce expression of FOXP3, the master transcription factor for tT(reg). Targeted insertion of a robust enhancer/promoter proximal to the first coding exon bypassed epigenetic silencing, permitting stable and robust expression of endogenous FOXP3. HDR-edited T cells, edT(regs), manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tT(reg). Both human and murine edT(regs) mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edT(reg) with robust in vitro and in vivo functional activity. Last, edT(reg) could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edT(reg) production may permit broad future clinical application.