CRISPR/Cas9-mediated Cxcr4 Disease Allele Inactivation for Gene Therapy in a Mouse Model of WHIM Syndrome

• A 2-step gene therapy protocol of autologous HSPC transplantation for WHIM syndrome by CRISPR/Cas9-mediated disease allele inactivation

• WHIM disease allele inactivated HSPCs have a strong selective advantage for durable hematopoietic reconstitution over WHIM cells

WHIM syndrome is an autosomal dominant immunodeficiency disorder caused by gain-of-function mutations in chemokine receptor CXCR4 that promote severe panleukopenia due to bone marrow (BM) retention of mature leukocytes. We previously reported that Cxcr4-haploinsufficient (Cxcr4^+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wildtype (Cxcr4^+/+) and WHIM (Cxcr4^+/w) HSCs, and that a WHIM patient was spontaneously cured by chromothriptic deletion of the disease allele in an HSC, suggesting that WHIM allele inactivation through gene editing may be a safe genetic cure strategy for the disease. We have now developed a two-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation towards this goal. First, one copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between WHIM and wildtype Cxcr4 alleles. Then, through in vivo natural selection, WHIM allele-inactivated cells were enriched over wildtype allele-inactivated cells. The WHIM allele-inactivated HSCs retained long-term pluripotency and selective hematopoietic reconstitution advantages. To our knowledge this is the first example of gene therapy for an autosomal dominant gain-of-function disease using a disease allele inactivation strategy in place of the less efficient disease allele repair approach.