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 p47^phox-deficient chronic granulomatous disease (p47-CGD) is a life-threatening immune deficiency caused by mutations in <i>NCF1</i>, a gene with 2 pseudogenes, <i>NCF1B</i> and <i>NCF1C</i>. The most common <i>NCF1</i> mutation, a GT deletion (ΔGT) at the start of exon 2 (>90% of alleles), is constitutive to <i>NCF1B</i> and <i>NCF1C</i>. <i>NCF1</i> ΔGT 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 ΔGT in induced pluripotent stem cells or CD34⁺ hematopoietic stem cells derived from p47-CGD patients. Correction of ΔGT in <i>NCF1</i> pseudogenes restores oxidase function in p47-CGD, providing the first demonstration that targeted restoration of pseudogene function can correct a monogenic disorder.