Modeling and correction of SCID-X1 using CRISPR-Cas9 homology-directed repair in human HSPCs.

X-linked severe combined immunodeficiency (SCID-X1) is a severe primary immunodeficiency caused by mutations in the IL2RG gene, a shared subunit of cytokine receptors critical for the development and function of T and natural killer (NK) cells. The standard treatment, allogeneic hematopoietic stem cell transplantation (HSCT), requires a compatible donor and is often associated with significant transplant-related complications. We aimed to develop a more robust and universal gene therapy by ex vivo modification of patient hematopoietic stem and progenitor cells (HSPCs) using the CRISPR-Cas9/rAAV6 gene-editing platform. To evaluate efficiency, we utilized a feeder-free, in vitro platform enabling T and NK cell differentiation from modified HSPCs. We investigated two approaches: the cut-site method (inserting a corrective cassette downstream of the start codon within the IL2RG locus) and a replacement method (replacing the entire IL2RG gene under endogenous regulation). We demonstrated that the cut-site insertion strategy is preferable for SCID-X1 correction, achieving superior homology-directed repair rates, lower toxicity, and significantly improved T and NK cell differentiation, based on phenotypic marker expression. Importantly, corrected patient-derived HSPCs from two SCID-X1 patients, modified using the cut-site approach, successfully demonstrated phenotypic evidence of T cell differentiation. Therefore, our findings firmly establish the feasibility of the cut-site strategy as a universal, high-efficiency, therapeutic solution for SCID-X1.