Functional analysis of myeloid cells generated from corrected homozygous exon 2 ΔGT NCF1 p47-CGD iPSCs. The cell treatment procedure is summarized above the graphs, with minigene insertion (ins) mediated by donor A or exon 2 replacement (GT ins) mediated by donor C. (A) Flow cytometric analysis of p47phox expression in myeloid-differentiated iPSC lines, P47-04 (uncorrected) vs minigene-corrected, or healthy control (left 3 panels, respectively) and in undifferentiated iPSCs (right 3 panels). (B) Oxidase function of the uncorrected, minigene-corrected, and healthy control line (left 3 panels, respectively) or the same iPSC lines without differentiation (right 3 panels, respectively) evaluated by dihydrorhodamine (DHR) flow cytometry analysis. (C) Flow cytometric analysis of p47phox expression in myeloid-differentiated iPSC lines, P47-04 (uncorrected) vs five exon 2–corrected, and healthy control, respectively. (D) Oxidase function of the uncorrected, 5 exon 2–corrected, and healthy control lines, respectively, or the same iPSC lines without differentiation, respectively, was evaluated by DHR. Also indicated below these graphs (Donor target) is where the correction occurred (NCF1 gene or the NCF1B or NCF1C pseudogene) as determined by sequence analysis (Figure 5). rAAV2 vector was used between 200 and 400 MOI of viruses per cell. SSC, side scatter; WT, wild-type.
