Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease

Abstract

Pseudogenes are duplicated genes with mutations rendering them nonfunctional. For single-gene disorders with homologous pseudogenes, the pseudogene might be a target for genetic correction. Autosomal-recessive p47phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in NCF1, a gene with 2 pseudogenes, NCF1B and NCF1C. The most common NCF1 mutation, a GT deletion (DGT) at the start of exon 2 (.90% of alleles), is constitutive to NCF1B and NCF1C. NCF1 DGT results in premature termination, undetectable protein expression, and defective production of antimicrobial superoxide in neutrophils. We examined strategies for p47-CGD gene correction using engineered zinc-finger nucleases targeting the exon 2 DGT in induced pluripotent stem cells or CD341 hematopoietic stem cells derived from p47-CGD patients. Correction of DGT in NCF1 pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.