Research ArticleGENE EDITING

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

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Science Translational Medicine  03 Jun 2020:
Vol. 12, Issue 546, eaay6422
DOI: 10.1126/scitranslmed.aay6422

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Enforced editing

Various autoimmune diseases could potentially be treated with regulatory T cells (Tregs), but there are many hurdles between this idea and clinical execution. Honaker et al. devised a gene-editing strategy to enforce expression of FOXP3, the master Treg transcription factor, in CD4+ T cells isolated from human peripheral blood, thereby overcoming limitations of Treg isolation and expansion. Resulting stable FOXP3 expression enabled a suppressive phenotype in vitro, and the edited cells were also functional in a xenogeneic graft-versus-host disease model and an experimental autoimmune encephalitis model. This approach has the potential to rapidly translate to clinical use.


Thymic regulatory T cells (tTregs) are potent inhibitors of autoreactive immune responses, and loss of tTreg function results in fatal autoimmune disease. Defects in tTreg number or function are also implicated in multiple autoimmune diseases, leading to growing interest in use of Treg as cell therapies to establish immune tolerance. Because tTregs 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 Treg-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 tTreg. 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, edTregs, manifested a transcriptional program leading to sustained expression of canonical markers and suppressive activity of tTreg. Both human and murine edTregs mediated immunosuppression in vivo in models of inflammatory disease. Further, this engineering strategy permitted generation of antigen-specific edTreg with robust in vitro and in vivo functional activity. Last, edTreg could be enriched and expanded at scale using clinically relevant methods. Together, these findings suggest that edTreg production may permit broad future clinical application.

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