Preclinical efficacy and safety assessment of engineered regulatory T cells for treatment of IPEX and other autoimmune disorders.

FOXP3 is an essential transcription factor driving lineage commitment and function of regulatory T cells (Tregs). We previously reported a proof-of-concept study describing engineered Tregs (EngTregs) generated using homology-directed repair-based editing of the FOXP3 gene in CD4+ T cells, resulting in constitutive, high-level endogenous FOXP3 expression leading to acquisition of a Treg phenotype and robust in vitro and in vivo suppressive function. Here, we expand this gene-editing strategy to integrate a functional FOXP3 cDNA to enable EngTreg therapy for immune dysregulation, poly-endocrinopathy, enteropathy, X-linked syndrome (IPEX), a devastating multiorgan autoimmune disorder mediated by mutations within the FOXP3 gene. We provide a detailed characterization of preclinical EngTreg generation, including gene targeting efficiencies, cell enrichment and expansion, analyses of potential off-target editing, and phenotypic and functional characterization, including in vitro suppression of effector T cells and in vivo reversal of graft-versus-host disease. Importantly, in parallel, we utilize syngeneic and humanized mouse models to demonstrate the safety of EngTregs in primary and secondary humoral responses and viral infection control, respectively. Our combined preclinical dataset strongly supports the efficacy and safety of EngTregs as a promising potential cellular therapeutic approach for IPEX and possibly other autoimmune disorders.