Conversion of T-Effector Cells to Immunosuppressive T-Regulatory-like Cells By CRISPR/Cas9-Mediated Integration of a FOXP3 Transgene

Regulatory T cells (Treg) are distinct among T cell subtypes, having the primary role of suppressing adaptive immune responses. The importance of these cells in immune self-tolerance is underscored by the genetically inherited syndrome IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), which is caused by an inactivating mutation in FOXP3. FOXP3 is a transcription factor that is a determinant of regulatory T cell function. Patients with IPEX syndrome suffer from the rapid and severe onset of multi-organ autoimmunity, including severe enteropathy, Type I diabetes, thyroiditis, skin inflammation and other features. In mouse models of IPEX, neonatal transplantation of wild-type Tregs is sufficient to prevent the development of disease. Less-severe Treg defects have also been implicated in the etiology of a variety of prevalent autoimmune diseases. It is possible that the pivotal role for Tregs in self-tolerance could be exploited clinically to improve therapies for autoimmunity and other diseases of tolerance. However, the use of autologous ex vivo expanded Treg as a clinical cell therapy is problematic: Tregs are present in low numbers in the peripheral blood, they expand slowly in culture ex vivo, and they may lack antigen specificities necessary for efficient suppression in specialized tissues. They may also down-regulate FOXP3 expression and lose functional activity in vivo in the setting of chronic inflammation. Additionally, autologous Tregs from patients with autoimmune disease may exhibit cell intrinsic dysfunction, while IPEX patients do not even have Tregs. To overcome these issues, we developed a gene editing approach to enforce stable expression of FOXP3 in primary human CD4⁺ peripheral blood T cells. CRISPR/Cas9 ribonucleoprotein and an AAV6-delivered donor template were developed to target a MND promoter-FOXP3 cDNA expression cassette (linked to a cell surface LNGFR tag by a 2A ribosome skip peptide) to the FOXP3 locus by homology directed repair (HDR). Highly efficient HDR rates were achieved across multiple donors (~34%; 5 donors in 9 experiments). For therapy of IPEX caused by FOXP3 missense mutations, integration of the functional coding sequence simultaneously abolishes endogenous FOXP3 expression. Following gene editing, expression of FOXP3 was sufficient to drive Treg-like phenotypic changes, including the up-regulation of CD25 and inhibitory receptors and down-regulation of CD127 and inflammatory cytokines. Further, consistent with the translatability of this approach into clinical manufacturing, FOXP3⁺ cells could be enriched to >90% purity by a simple LNGFR antibody column and expanded 20-fold within one week. Importantly, transfer of these edited Treg-like cells (edTreg) to NOD-scid-IL2Rγ-/- mice prevented xeno-graft vs. host disease (xeno-GvHD) mediated by co-transferred autologous effector T cells; xeno-GvHD protection correlated with long-term survival of the edTregs, and a marked reduction in effector T cell expansion and tissue infiltration. These data support the development of edited regulatory T cells for the treatment of IPEX and other autoimmune disease.