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 because of retention of mature leukocytes in the bone marrow (BM). We previously reported that Cxcr4-haploinsufficient (Cxcr4^+/o) hematopoietic stem cells (HSCs) have a strong selective advantage for durable hematopoietic reconstitution over wild-type (Cxcr4^+/+) and WHIM (Cxcr4^+/w) HSCs and that a patient with WHIM 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 developed a 2-step preclinical protocol of autologous hematopoietic stem and progenitor cell (HSPC) transplantation to achieve this goal. First, 1 copy of Cxcr4 in HSPCs was inactivated in vitro by CRISPR/Cas9 editing with a single guide RNA (sgRNA) that does not discriminate between Cxcr4^+/w and Cxcr4^+/+ alleles. Then, through in vivo natural selection, WHIM allele–inactivated cells were enriched over wild-type 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.