332. ZFN-Mediated Minigene or Dinucleotide Gene Correction of p47phox Deficient Autosomal Recessive Chronic Granulomatous Disease iPSC to Generate Oxidase Functional Neutrophils

Chronic granulomatous disease is an immune deficiency caused by defective phagocyte NADPH oxidase resulting in severe infections and autoimmune complications. NCF1 gene mutations resulting in p47phox deficiency are the most common cause of autosomal-recessive chronic granulomatous disease (AR-CGD), and notably, >80% of patients have homozygous GT-deletions in exon 2, resulting in a frame shift, premature termination, and absence of p47phox expression. Here we show two nucleofection strategies for NCF1 gene correction using zinc-finger nuclease (ZFN) targeting the NCF1 locus in patient EBV-immortalized B cells and in induced pluripotent stem cells (iPSC) derived from p47phox-deficient patients. With the first strategy, ZFN-mediated cut at start of exon 2 allows insertion of a minigene (NCF1 cDNA with deleted exon 1) to allow continuous transcription from the endogenous NCF1 exon 1 to the donor minigene exon 2 until the end of the cDNA and beta-globin polyadenylation signal. The second strategy takes advantage of the most prevalent GT-deletion by providing a dinucleotide corrective donor plasmid for gene editing using the same ZFNs. The two donors contain ~750 bp to 1000 bp flanking homology arms and also encode a selectable marker: a puromycin resistance gene flanked by loxP sites to allow for excision after selection. In patient EBV-immortalized B cells, minigene and dinucleotide targeted gene correction of constitutively active NCF1 was 8-20% after puromycin-selection from obtained clones. The rates of correction in iPSC were generally lower, less than 2%. Use of AAV2 vector to deliver dinucleotide corrective donor to iPSC increased insertion efficiency to >80% after puromycin selection. Gene corrected B cells for both correction strategies restored ROS function, shown by chemiluminescence assay. Neutrophils differentiated from gene corrected iPSCs demonstrated correction of oxidase activity by chemiluminescence or dihydrorhodamine flow cytometry assay. With both methods, p47phox protein expression is seen in neutrophils differentiated from iPSC but not in undifferentiated iPSC, demonstrating cell type-specific regulation of expression at the corrected locus. In summary, we show that iPSC generated from CGD patient cells can be gene corrected through a targeted dinucleotide correction method for greater than 80% of alleles and minigene correction for mutations other than the GT-deletions.