Targeted Repair of p47-CGD Restores the Ability of iPSC-Derived Macrophages to Kill Bacteria

Chronic granulomatous disease (CGD) is caused by mutations in the NADPH oxidase and is characterized by defective phagocytes. The NAPDH oxidase is important for the production of reactive oxygen species that further regulate the phagosome milieu, such as the release of granules containing antimicrobial proteins, and the killing of bacteria inside the phagosome, which is defective in CGD patients. Only allogenic hematopoietic stem cell transplantation (HSCT) and retroviral gene therapy are curative treatment options for CGD patients. The first gene therapy trials for CGD were complicated by insertional mutagenesis and transgene silencing of the applied LTRdriven gamma-retroviral vector. More promising results were shown by recent studies with lentiviral SIN vectors and a myeloid-specific promoter. However, targeted gene correction using CRISPR-Cas9 would avoid the risk of vector-mediated insertional mutagenesis and silencing of the therapeutic gene. In our study, we developed a gene editing approach to correct p47phox-deficiency, which affects about25% of CGD patients. Over 90% of these patients carry the mutation c.75_76delGT (DGT) in exon 2 of NCF1 (encodes p47phox). This highprevalence most likely originates via gene conversion from one of the two pseudogenes NCF1B/C, which are over 99% homologous to NCF1 and carry the aforementioned DGT mutation. For proof-of-principle, we corrected p47phox-deficient induced pluripotent stem cells (iPSC)by inserting a minigene donor construct into intron 1 of NCF1. We chose this position, which differs by three additional nucleotides from the pseudogenes, to avoid cleavage in the pseudogenes and, thus, potential chromosomal instability due to multiple DNA doublestrand breaks. To analyze functionality, genetically corrected clones were differentiated into granulocytes and macrophages. Corrected granulocytes expressed p47phoxsimilar to wild type levels, displayedNADPH oxidase activity and were able to form neutrophil extracellular traps, which enable granulocytes to kill microbes extracellularly. Finally, iPSC-derived macrophages were infected with GFP-labeled E. coli. After phagocytosis, corrected macrophages significantly reduced the amount of living bacteria inside their phagosomes as measured by colony-forming units in plated cell lysates compared to uncorrected macrophages. In summary, we demonstrated that the specific insertion of a minigene into intron 1 of NCF1 corrects p47phox-deficiency.Moreover, the pseudogenes remained intact with our strategy, which could be of importance as the pseudogenes might have functional roles. We showed that the corrected iPSC-derived granulocytes and macrophages have all the features necessary to fight infections. In the past, some patients benefited from allogenic granulocyte transfusions. However, due to alloimmunization, this therapy is contraindicative for subsequent HSCT. We propose that autologous iPSC-derived granulocytes and macrophages could be applied therapeutically in the future to combat refractory infections prior to HSCT.