Gene editing using CRISPR enables FOXP3 gene repair in HSPCs and IPEX patient T cells

Abstract

Background & Aim Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is the prototypical primary immune regulatory disorder caused by mutations in the forkhead box protein 3 (FOXP3) gene, a critical transcription factor required for regulatory T cells (Tregs). Patients typically present with severe early onset autoimmune manifestations that can be fatal within the first year of life. Currently, the only cure for IPEX is allogeneic hematopoietic stem cell transplant (HSCT), but this comes with risk of complications and the survival probability at 15 years is 73.2% (F. Barzaghi et al 2018). Moreover, a suitable donor is not always available. As a monogenic immune disease with limited treatment options, IPEX is an ideal candidate for a gene therapy approach whereby patient hematopoietic stem cells are gene edited and reinfused for autologous transplant. Methods, Results & Conclusion We developed a CRISPR/Cas9 approach combined with AAV delivery of a donor template to restore FOXP3 expression at the endogenous locus, permitting regulated expression of wild-type FOXP3 irrespective of downstream mutations. We were able to edit CD4+ effector T cells (Teff) and Tregs, showing that they maintain characteristic phenotypic markers and are functional in vitro. IPEX patient Tregs edited with the construct had partial restoration of both FOXP3 expression and functional suppression upon co-culture with responder T cells. Gene edited cord blood hematopoietic stem and progenitor Cells (HSPCs) are capable of long-term engraftment in humanized mice and differentiate into multiple hematopoietic subsets (CD13, CD19, CD56, CD3) and partially reconstitute Tregs (CD4+CD25+FOXP3+). These results demonstrate the feasibility of using gene edited HSPCs and hold promise for autologous transplant in genetic autoimmune diseases such as IPEX syndrome.