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Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic disorder caused by mutations in the FOXP3 gene, required for generation of regulatory T (T reg ) cells. Loss of T reg cells leads to immune dysregulation characterized by multi-organ autoimmunity and early mortality. Hematopoietic stem cell (HSC) transplantation can be curative, but success is limited by autoimmune complications, donor availability and/or graft-vs.-host disease. Correction of FOXP3 in autologous HSC utilizing a homology-directed repair (HDR)-based platform may provide a safer alternative therapy. Here, we demonstrate efficient editing of FOXP3 utilizing co-delivery of Cas9 ribonucleoprotein complexes and adeno-associated viral vectors to achieve HDR rates of >40% in vitro using mobilized CD34 + cells from multiple donors. Using this approach to deliver either a GFP or a FOXP3 cDNA donor cassette, we demonstrate sustained bone marrow engraftment of approximately 10% of HDR-edited cells in immune-deficient recipient mice at 16 weeks post-transplant. Further, we show targeted integration of FOXP3 cDNA in CD34 + cells from an IPEX patient and expression of the introduced FOXP3 transcript in gene-edited primary T cells from both healthy individuals and IPEX patients. Our combined findings suggest that refinement of this approach is likely to provide future clinical benefit in IPEX., Competing Interests: S.S., I.F.K, Y.H., K.M.S., and D.J.R hold provisional patent PCT WO2019210042 entitled “Expression of FOXP3 in edited CD34+ cells” related to this work. Another patent, “Expression of Human FOXP3 in Gene Edited T Cells,” has also been filed with Y.H., K.M.S., I.F.K, and D.J.R. as authors., (© 2023 The Author(s).)

Adoptive transfer of regulatory T cells (T regs ) is therapeutic in type 1 diabetes (T1D) mouse models. T regs that are specific for pancreatic islets are more potent than polyclonal T regs in preventing disease. However, the frequency of antigen-specific natural T regs is extremely low, and ex vivo expansion may destabilize T regs , leading to an effector phenotype. Here, we generated durable, antigen-specific engineered T regs (EngT regs ) from primary human CD4 + T cells by combining FOXP3 homology-directed repair editing and lentiviral T cell receptor (TCR) delivery. Using TCRs derived from clonally expanded CD4 + T cells isolated from patients with T1D, we generated islet-specific EngT regs that suppressed effector T cell (T eff ) proliferation and cytokine production. EngT regs suppressed T effs recognizing the same islet antigen in addition to bystander T effs recognizing other islet antigens through production of soluble mediators and both direct and indirect mechanisms. Adoptively transferred murine islet-specific EngT regs homed to the pancreas and blocked diabetes triggered by islet-specific T effs or diabetogenic polyclonal T effs in recipient mice. These data demonstrate the potential of antigen-specific EngT regs as a targeted therapy for preventing T1D.