Genome editing using the CRISPR-Cas9 system to correct IL2RG-SCID mutations in Hematopoietic stem and progenitor cells

The immune system is composed of two subsystems – adaptive and innate - that work in harmony to protect the human body from infections and diseases. A defect in T, B and Natural Killer (NK) cells affects both the adaptive and innate systems and therefore can lead to Severe Combined Immunodeficiency (SCID). The most common type of SCID worldwide is X-SCID, caused by mutations in the IL2RG gene. Since IL2RG is a shared subunit of 6 different cytokine receptors critical for various stages of T and NK cell differentiation and functionality, the resulting phenotype is the absence of T and NK cells. Genome editing of hematopoietic stem and progenitor cells (CD34+ HSPCs) could provide a therapeutic solution for SCID patients. In this study we have developed a therapeutic approach based on CRISPR-Cas9 genome editing to correct disease-causing mutations in the IL2RG gene by precisely engineering the CD34+ HSPCs. We have established a genome editing strategy in which we used CRISPR-Cas9 together with rAAV6 donor DNA template to replace the IL2RG gene with a corrective cDNA. Using novel feeder-free In Vitro T and NK cell differentiation assays, we have demonstrated that our system can simulate and model the correction process and that our cDNA supports the successful differentiation of both T and NK cells. In conclusion, our study demonstrates the potential of CRISPR-Cas9 genome editing for X-SCID gene correction and possibly other hematopoietic disorders.