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

Abstract

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.

IPEX as a Consequence of Alternatively Spliced FOXP3

“…Essential for this strategy is the targeting of splice-shifting elements that has shown promising potential in other isoform-related diseases (90,91). Alternatively, also gene editing that enhances FOXP3fl expression may provide a new approach to regulate human T-cell responses (76,92).…”

“…However, it has become clear that identical FOXP3 mutations can cause different disease manifestation in IPEX patients(69). This reflects the stochastic nature of autoreactive responses [reviewed inRichards et al This notion is corroborated by the recent finding that T cells with enforced expression of FOXP3 c.239del via gene editing do not suppress T-cell proliferation(76). Thus, absence of FOXP3fl prevents phenotypical and functional Treg-cell development and FOXP3 2 expression alone in IPEX patients with FOXP3 exon 2 mutations does not characterize bona fide…”

Super-Treg: Toward a New Era of Adoptive Treg Therapy Enabled by Genetic Modifications

“…This strategy could also allow high numbers of Treg to be obtained from Tconv. In CD4 + Tconv, ectopic expression of FOXP3 using retroviral vectors ( 22 , 26 , 43 ) or by HDR of a strong promoter upstream of the FOXP3 coding sequences ( 19 ) allowed generation of Treg that suppressed CD4 + Tconv not only in vitro but also inhibited GvHD, colitis or dermatitis in animal models ( Table 1 ) . Interestingly, tamoxifen-induced but not constitutive FOXP3 expression in CD4 + Tconv resulted in control of autoimmune arthritis by migration into lymph nodes ( 39 ).…”

New Frontiers about the Role of Human Microbiota in Immunotherapy: The Immune Checkpoint Inhibitors and CAR T-Cell Therapy Era

“…Present in very small quantities in peripheral blood and physiologically characterized by powerful inhibitor action on autoreactive immune responses, the tT regs have a powerful function to prevent the activation of autoimmune response. Using CRISPR-Cas9 system, these scientists created autologous T reg -like cells amplifying the expression of the master transcription factor for tT regs FOXP3, inserting a powerful enhancer/promoter to its first coding exon, thus leading immunosuppression in vivo human and murine models of the disease [ 11 ].…”