Volume 23, Issue 1, Pages 147-157 (January 2015) An AAVS1-Targeted Minigene Platform for Correction of iPSCs From All Five Types of Chronic Granulomatous Disease  Randall K Merling, Colin L Sweeney, Jessica Chu, Aaron Bodansky, Uimook Choi, Debra Long Priel, Douglas B Kuhns, Hongmei Wang, Sam Vasilevsky, Suk See De Ravin, Thomas Winkler, Cynthia E Dunbar, Jizhong Zou, Kol A Zarember, John I Gallin, Steven M Holland, Harry L Malech  Molecular Therapy  Volume 23, Issue 1, Pages 147-157 (January 2015) DOI: 10.1038/mt.2014.195 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Targeted gene correction and transgene expression in iPSC. (a) Schema of AAVS1-targeted gene correction strategy in CGD patient iPSCs co-transfected with AAVS1 ZFNs and one of five targeting donor minigene plasmids (CGD donor). The donor plasmids (shown here as a linear construct) contain AAVS1 region homology arms (800 base pairs each) homologous to the regions flanking the AAVS1 ZFN target site on chromosome 19, resulting in targeted insertion of the donor plasmid transgenes upon cutting of that site by the ZFNs. A splice acceptor site is located upstream of the puromycin resistance gene (with a bovine growth hormone polyadenylation signal), so that puromycin will be expressed when the splice acceptor site can capture expression from the AAVS1 site (PPP1R12C gene). An internal cytomegalovirus early enhancer element with a chicken β actin promoter (CAG) is used to express the therapeutic phox cDNA minigene (with a rabbit β-globin polyadenylation signal). Locations of primers for insertion site analyses are shown (arrowheads): white denotes AAVSCEL primers for the untargeted AAVS1 locus, gray denotes AAVS1U-F and PuroU-R primers for targeted insertion, and black denotes R-Random and L-Random primers for random insertion. Locations of Southern blot probe and SphI restriction sites are also shown. (b) Constitutive AAVS1-phox transgene expression in CGD iPSCs after targeted gene insertion. CGD patient iPSC lines were gene-corrected with the appropriate CGD donor construct according to the strategy described above, resulting in constitutive expression of that phox transgene from the CAG promoter, as demonstrated here by flow cytometry analysis with the respective antibody staining. The corresponding uncorrected iPSC line is shown in black and corrected cell lines are shaded gray. (c) Southern blot analysis of clones pre-screened for targeted insertion at the AAVS1 locus by PCR. Genomic DNA was digested with SphI restriction enzyme and probed with radiolabeled puromycin-resistance gene. Lane numbers correspond to the following samples: 1 (GeneRuler ladder), 2 (iP47-Uncorrected), 3 (iP47-AAVS1 c1), 4 (iP47-AAVS1 c2), 5 (GeneRuler ladder), 6 (iP40-Uncorrected), 7 (iP40-AAVS1 c1), 8 (iP67-Uncorrected), 9 (iP67-AAVS1 c3), 10 (iGP91-Uncorrected), 11 (iGP91-AAVS1 c1), 12 (GeneRuler ladder), 13 (iP40-AAVS1 c3), 14 (iP67-AAVS1 c6), 15 (iP67-Uncorrected), and 16 (iP67-AAVS1 c10). Shown are 10, 5, and 4 kb DNA fragment sizes. A single band around 3.8 kb indicates targeted homologous recombination at the AAVS1 locus. Off-target insertion of the intact plasmid vector results in a slightly larger 4.3 kb band, as seen in some clones. (d) PCR analysis for off-target vector insertions based on the presence of plasmid backbone sequences outside of the left homology arm region (LHA) and right homology arm (RHA) region, for clones pre-screened for targeted insertion at the AAVS1 locus. Lane numbers correspond to the following samples: 1 (1 kb ladder), 2 (iP22-AAVS1 c2), 3 (iP22-AAVS1 c3), 4 (iP22-AAVS1 c4), 5 (iP22-AAVS1 c11), 6 (1 kb ladder), 7 (iGP91-Uncorrected), 8 (iGP91-AAVS1 c1), 9 (iGP91-AAVS1 c2), 10 (iGP91-AAVS1 c3), 11 (iGP91-AAVS1 c4), 12 (iGP91-AAVS1 c5), 13 (iGP91-AAVS1 c6), and 14 (iGP91-AAVS1 c7). Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Pluripotency characterization of iPSCs after genetic correction. (a) Gene-corrected iPSC colonies were observed by bright field and fluorescence microscopy for alkaline phosphatase live stain (green) and TRA-1–60 antibody stain (red). The representative sample shown is from iP40-AAVS1 (the corrected iP40-01 cell line). Scale bar = 500 μm. (b) Teratoma containing all three germ layers: cartilage (mesoderm), neural crest (ectoderm), and epithelial tissue (endoderm). The representative sample shown is from iP67-02-AAVS1. Scale bar = 500 μm. (c) G-band karyotype of corrected iP40-AAVS1 iPSCs. Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Schematic diagram of APEL hematopoietic differentiation of iPSCs with additional downstream differentiations. The different media changes are described over the days of the differentiations, with APEL based medium used for Media A and B. After 7 days expansion of CD34+ cells in stage 3, the final differentiation is performed using G-CSF to obtain neutrophils or M-CSF to obtain macrophages. Cryopreservation can be done near the end of stage 3 and after macrophage differentiation. Functional macrophages are derived from 3 days culture with IFN-γ. Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Characterization of myeloid cells differentiated using G-CSF from iPSCs from all CGD corrections. AAVS1 gene-corrected from parental CGD iPSCs lines were differentiated into neutrophils using G-CSF and analyzed for identity by (a) Giemsa stain of cytospun cells (scale bar = 20 μm) and (b) analysis of neutrophil surface markers. The unshaded black histogram represents the isotype control and the shaded gray histogram is the fluorescent antibody staining for CD11b, CD13, CD15, CD16b, and CD33. (c) DHR flow cytometric analysis of granulocytes. The upper row of panels of 4c from left to right shows: analysis of a differentiated representative CGD uncorrected iPSC line; normal donor peripheral blood; a differentiated representative normal control iPSC line; and differentiated AAVS1-targeted gene-corrected p40phox, p67phox, and gp91phox iPSC lines, respectively. The lower panel shows analysis for differentiated uncorrected CGD iPSC; differentiated normal control iPSC; and AAVS1-targeted gene-corrected p22phox and p47phox iPSC lines, respectively. The gated region in each sample encompasses the mean fluorescence of normal granulocyte controls analyzed in the same experiment. The number in the gate indicates the percent of DHR positive granulocytes within the total cell population (consisting of granulocytes, monocyte/macrophages, and in some differentiations, immature cells). (d) Enhanced chemiluminescence of neutrophils differentiated from uncorrected (left) or corrected (right) iPSCs stimulated with PMA. The numbers represent the area under the curve of chemiluminescence emission, measured over 40 minutes. Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Characterization of macrophages differentiated from corrected iPSC (iP22-AAVS1). Shown are M-CSF derived macrophages analyzed by (a) cytospin Giemsa stain for macrophage morphology (scale bar = 50 μm) and (b) flow cytometry for macrophage cell surface markers CD14 and CD163 and the hematopoietic marker CD45. (c) Functional analysis of respiratory oxidative burst activity by the DHR assay. Shown here are representative examples of uncorrected (left) and corrected (right) macrophages differentiated from iP22-01 iPSCs, demonstrating ROS production by the corrected macrophages. (d) ELISA assay of IL-10 production by macrophages in response to UV-inactivated Listeria. Peripheral blood monocyte derived macrophages (MDM) and corrected-iPSC derived macrophages (iPSC-MO) were incubated with media only (Media) or infected with Listeria (Lm). Supernatant was harvested and tested for IL-10. Data points were plotted from six independent experiments of corrected (iP22-AAVS1 and iP47-AAVS1) M-CSF derived macrophages and four independent experiments from different normal donor MDMs. Error bars denote mean ± SD. M-CSF differentiated macrophages derived from iPSC showed a significant increase in IL-10 production (P = 0.0007, by Mann–Whitney nonparametric test) in response to UV-inactivated Listeria. Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Antimicrobial activity of iPSC-derived neutrophils or macrophages. (a) Staphylocidal activity of neutrophils derived from uncorrected (iP22) and corrected CGD iPSCs (iP22-AAVS1 and iP67-AAVS1), from three independent experiments. Controls include media only (without cells) and normal donor peripheral blood neutrophils. Data are represented as percent bacteria surviving after a 20-minute infection period at an MOI of 2, demonstrating antimicrobial activity by neutrophils differentiated from corrected iPSCs, to levels comparable to normal blood neutrophils. Error bars denote mean ± SD. (b) M-CSF macrophages derived from uncorrected (-) and AAVS1 gene-corrected CGD iPSCs (+) or monocyte-derived macrophages (MDM) from normal (n = 6) and CGD (n = 7) donors were treated with IFN-γ 2–3 days before infection with Granulibacter at an MOI of 1 for 24 hours. Macrophages were lysed and bacteria enumerated after dilution plating. Uncorrected and corrected iPSC-derived cells were tested in up to seven independent experiments: iP22 (n = 7; P = 0.016); iP67 (n = 5); iP40 (n = 1); iP47 (n = 1). Error bars denote mean ± SD. For comparison of differentiated macrophages from all uncorrected versus corrected iPSC lines, P = 0.0001 (***); for normal versus CGD MDM, P = 0.001 (**). Statistical analysis was performed using Mann–Whitney nonparametric test (normal versus CGD) or Wilcoxon nonparametric test (uncorrected versus corrected iPSC). Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 A schematic diagram for generating iPSCs from CGD patient peripheral blood CD34+ cells, followed by genetic correction. Five CGD types are shown in which CD34+ cells from peripheral blood are reprogrammed to iPSCs and then gene-corrected in a platform for genetic correction using targeted homologous recombination genetic correction via AAVS1 site specific ZFNs. This diagram also depicts the five major phox subunits in the active form of NADPH oxidase where electron transport occurs to form superoxide anion and hydrogen peroxide, as in phagosomes containing ingested microorganisms. Molecular Therapy 2015 23, 147-157DOI: (10.1038/mt.2014.195) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions