IL10 gene mutations do not impair Tr1 differentiation in vivo and can be functionally corrected by precise gene editing 9296

Abstract Description IL10 deficiency is a life-threatening form of IBD that is difficult to treat. IL-10 is a potent anti-inflammatory cytokine secreted by various cell types, including immunosuppressive CD4+ Type 1 regulatory T (Tr1) cells, which can be generated in vitro in the presence of IL-10. To investigate whether Tr1 cells are present in patients with IL10 deficiency, we characterized the PBMC immune phenotype of two symptomatic IL10-deficient patients and detected normal percentages of CD49b+LAG-3+ Tr1 cells. Both patient-derived Tr1 cell clones and IL10RA-deficient Tr1 cells, generated using CRISPR/Cas9, showed suppressive capacity towards CD4+ T cells in vitro, which was partially reversed by blocking CTLA-4. Importantly, the absence of pSTAT-3 upregulation in responder CD4+ T cells confirmed the mutant IL-10 was dysfunctional. One of the patients underwent a matched unrelated donor hematopoietic stem cell transplantation (HSCT) with stable and selective long-term engraftment of the donor CD3+ T cells, but not the myeloid cells, suggesting that a wild-type T cell compartment is sufficient for clinical remission and disease control. We inserted a functional copy of the IL10 gene in CD4+ T cells using CRISPR/Cas9 and rAAV6 and observed physiological IL-10 secretion. In addition, CD34+ HSCs harboring this IL10 construct engraft and differentiate normally in NSGS humanized mice. Overall, IL10 gene editing of T cells or HSCs could be a promising therapeutic approach for IL10 deficiency. Funding Sources Maternal and Child Health Research Institute (MCHRI), Stanford University Innovative Medicines Accelerator (IMA), Stanford University Center for Genetic Immune Disease (CGID), Stanford University Topic Categories Therapeutic Approaches to Autoimmunity (THER)