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.

胸腺调节性T细胞（tT _regs）是自身反应性免疫反应的有效抑制剂，tT _reg功能丧失会导致致命的自身免疫性疾病。tT _reg数目或功能的缺陷也与多种自身免疫性疾病有关，导致越来越多的人将T _reg用作细胞疗法来建立免疫耐受性。由于tT _regs在循环血液中的数量很少，在某些受试者中可能难以纯化和扩增，并且固有地存在缺陷，因此我们设计了另一种策略，可从大量CD4 ⁺产生自体T _reg样细胞T细胞。我们使用了基于同源直接修复（HDR）的基因编辑来增强FOXP3（tT _reg的主要转录因子）的表达。在第一个编码外显子附近有针对性地插入稳健的增强子/启动子，绕过了表观遗传沉默，使内源性FOXP3能够稳定，稳定地表达。HDR编辑的T细胞edT _regs表现出转录程序，导致规范标记物的持续表达和tT _reg的抑制活性。人和鼠的EDT_的REG在炎性疾病模型介导的免疫体内。此外，该工程策略允许产生抗原特异性edT _reg具有强大的体外和体内功能活性。最后，可以使用临床相关方法扩大edT _reg的规模并扩大规模。在一起，这些发现表明edT _reg生产可能允许广泛的未来临床应用。