A Platform Minigene AAVS1 Targeted Safe Harbor Approach For Genetic Correction Of iPSC Derived From Patients With Each Of The 5 Genetic Forms Of Chronic Granulomatous Disease

There are 5 genetic forms of chronic granulomatous disease (CGD), resulting from mutations in one of the CYBB, CYBA, NCF1, NCF2, or NCF4 genes encoding, respectively, the subunits of the phagocyte oxidase (phox) gp91phox, p22phox, p47phox, p67phox and p40phox. Phox is required for blood neutrophils and monocytes to produce microbicidal superoxide and hydrogen peroxide. We previously demonstrated zinc finger nuclease (ZFN) targeting of a corrective minigene encoding gp91phox to the AAVS1 safe harbor site of iPSC from a patient with X-linked CGD (XCGD), which restored phox activity in neutrophils differentiated from the corrected iPSC (Blood 2011, 117:5561). Subsequently, we have generated iPSC from the CD34⁺ hematopoietic stem cells (HSC) present in small volumes of peripheral blood from additional CGD patients to include those representing the other genetic forms of CGD (Blood 2013, 121:e98). We used our previously reported gp91phox AAVS1 targeting plasmid to create CGD genotype-specific targeting plasmids for correction of the other 4 genetic forms of CGD by replacing gp91 cDNA with cDNA for each of the autosomal recessive CGD genes. Using these genotype specific targeting plasmids with AAVS1 targeting ZFNs, this approach resulted in safe harbor minigene correction of iPSC for each of the 5 genetic forms of CGD, routinely achieving minigene insertion with constitutive expression of the specific desired transgene phox subunit protein in 70-80% percent of selected iPSC clones. Neutrophils or macrophages were generated from iPSC by first differentiating in STEMdiff APEL medium for 13 days to generate CD34⁺CD45⁺ cells, which were further expanded in HSC medium for 7 days. The expanded cells were differentiated either to neutrophils with G-CSF in the presence or absence of OP9 mouse stromal cells for 7 days, or to macrophages with M-CSF for 10 to 14 days. Full restoration of oxidase activity was observed in neutrophils or macrophages derived from minigene corrected CGD iPSC representing all the 5 genetic types of CGD. Furthermore, we demonstrated significant correction of bacterial growth inhibition function in corrected CGD iPSC derived neutrophils (Staphylococci) and macrophages (Granulibacter). Of particular note, the M-CSF differentiated macrophages derived from iPSC showed a significant increase in IL-10 production (p < 0.01) in response to UV-inactivated Listeria, with comparable levels to M-CSF derived macrophages differentiated from peripheral blood monocytes. This type of iPSC gene-correction approach with ex vivo production of autologous functional neutrophils or macrophages could be used for supportive autologous cell therapy in CGD patients with infections not responding to conventional treatment. Using a universal platform approach that combines iPSC generation from small volumes of peripheral blood and ZFN mediated AAVS1 safe harbor minigene targeting, we show it is possible to efficiently go from peripheral blood sample to genetically corrected iPSC to generation of functionally corrected neutrophils and macrophages using an identical approach for all 5 genetic forms of CGD. This platform approach is broadly applicable to a wide range of inherited single gene metabolic disorders.