57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs
Authors: Colin L. Sweeney, Jizhong Zou, Uimook Choi, Randall K. Merling, Suk See DeRavin, Harry L. Malech
Journal: Molecular Therapy (2015)

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted "safe-harbor" gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with "excision-only" piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.