Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47ᵖʰᵒˣ-deficient chronic granulomatous disease

Abstract

p47ᵖʰᵒˣ-deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47ᵖʰᵒˣ expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67ᵖʰᵒˣ, an NADPH oxidase subunit that closely interacts with p47ᵖʰᵒˣ and is predominantly expressed in myeloid cells. This approach restored p47ᵖʰᵒˣ expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47ᵖʰᵒˣ-deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47ᵖʰᵒˣ and p67ᵖʰᵒˣ, ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.