Mol Ther. 2026 Mar 13. pii: S1525-0016(26)00202-9. [Epub ahead of print]
Yuchi Honaker,
Noelle P Dahl,
Swati Singh,
Akhilesh K Singh,
Christina Lopez,
David Gruber,
Samuel C Scharffenberger,
Shivani Patel,
Harold Jones,
Iram F Khan,
Michelle L Christian,
Su Jung Yang,
Karen Sommer,
Troy R Torgerson,
Eric J Allenspach,
Cade Ellis K Ito,
Gene I Uenishi,
Gregory J Cost,
Peter J Cook,
David J Rawlings.
FOXP3 is an essential transcription factor driving lineage commitment and function of regulatory T cells (Treg). We previously reported a proof-of-concept study describing engineered Treg (EngTreg) generated using HDR-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 detailed characterization of pre-clinical 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 GvHD. Importantly, in parallel, we utilize syngeneic and humanized mouse models to demonstrate the safety of EngTreg in primary and secondary humoral responses and viral infection control, respectively. Our combined pre-clinical dataset strongly supports efficacy and safety of EngTreg as a promising potential cellular therapeutic approach for IPEX and, possibly, other autoimmune disorders.