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

May 25, 2026Molecular therapy. Nucleic acids

Using CRISPR gene editing to fix SCID-X1 in human blood stem cells

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Abstract

The cut-site insertion strategy achieved superior homology-directed repair rates and significantly improved T and NK cell differentiation in modified patient hematopoietic stem and progenitor cells.

X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the IL2RG gene, affecting T and NK cell development.
Current treatment options like HSCT often require compatible donors and can lead to serious complications.
The CRISPR-Cas9/rAAV6 gene-editing platform was used to modify patient cells for potential gene therapy.
Two gene modification approaches were evaluated: cut-site insertion and complete gene replacement.
The cut-site method showed lower toxicity and better outcomes in T and NK cell differentiation compared to the replacement method.
Modified cells from two SCID-X1 patients exhibited phenotypic evidence of T cell differentiation using the cut-site approach.

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X-linked severe combined immunodeficiency (SCID-X1) is a severe primary immunodeficiency caused by mutations in thegene, 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 bymodification of patient hematopoietic stem and progenitor cells (HSPCs) using the CRISPR-Cas9/rAAV6 gene-editing platform. To evaluate efficiency, we utilized a feeder-free,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 thelocus) and a replacement method (replacing the entiregene 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. IL2RG ex vivo in vitro IL2RG IL2RG