CRISPR-targeted MAGT1 insertion restores XMEN patient hematopoietic stem cells and lymphocytes.

'X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus-infection and N-linked glycosylation defect' (XMEN) disease is a recently described primary immunodeficiency marked by defective T and Natural Killer (NK) cells. Potentially curative hematopoietic stem cell transplant is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for XMEN patients using CRISPR/Cas9/adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9/AAV-targeted gene editing (GE) is hampered by low engraftable GE hematopoietic stem/progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1-glycosylation function in human NK and CD8+ T cells restored NKG2D expression and function in XMEN lymphocytes for potential treatment of infections, and corrected HSPCs for long-term gene therapy, thus offering two efficient therapeutic options for XMEN poised for clinical translation.