Gene-edited pseudogene resurrection corrects p47phox-deficient chronic granulomatous disease by Randall K. Merling, Douglas B. Kuhns, Colin L. Sweeney, Xiaolin Wu, Sandra Burkett, Jessica Chu, Janet Lee, Sherry Koontz, Giovanni Di Pasquale, Sandra A. Afione, John A. Chiorini, Elizabeth M. Kang, Uimook Choi, Suk See De Ravin, and Harry L. Malech BloodAdv Volume 1(4):270-278 January 10, 2017 ©2017 by American Society of Hematology

Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Schematic diagram of NCF1 and its pseudogenes on chromosome 7 and the corrective donors used. Schematic diagram of NCF1 and its pseudogenes on chromosome 7 and the corrective donors used. (A) Positions of pseudogenes (NCF1B and NCF1C) relative to NCF1 are shown in the inset of chromosome locus 7q11.23. (B) Distinguishing sequences in normal NCF1 and pseudogenes include unique intronic SNPs, the normal GTGT (or mutant ΔGT) at start of exon 2 in NCF1 vs constitutive ΔGT in both NCF1B and NCF1C as depicted. We do not indicate in this figure that a portion of pseudogene or an entire pseudogene may replace a portion of or the entire NCF1 gene at its NCF1 locus. ZFN targets the start of exon 2, with the recognition sequence (upper case) and the spacer or cutting region (lower case) as shown. The ZFN targets the pseudogene sequence shown: CCCAGGTACATGTTCctggtgAAATGGCAGGAC. The capital letters represent the target sequence, and the lowercase letters denote the space or cutting site. Within the “Donor repair sequence,” the underlined base pair G was changed from a base pair A to avoid recutting but does not change the codon translation and is the codon-optimized choice. (C) Schematic representation of the correction donors used for minigene addition or exon 2 replacement with or without a puromycin (Puro) selection cassette flanked with loxP (A/B or C/D, respectively). The cytomegalovirus (CMV) promoter is used to express the puromycin resistance gene and the polyadenylation signal (pA) is indicated. Shown are left and right homology arms (LHA and RHA, respectively). The inverted terminal repeats (ITRs) in donors B, C, and D allow donor packaging in rAAV vector. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Functional analysis of myeloid cells generated from corrected homozygous exon 2 ΔGT NCF1 p47-CGD iPSCs. 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. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Oxidase activity following Cre excision. Oxidase activity following Cre excision. Analysis of DHR oxidase activity in myeloid differentiated iPSC lines following Cre-mediated recombination excision of the puromycin cassette. Shown are analyses of corrected iPSC lines (P47-04C2,4,5-Cre GT exon 2 corrected; P47-04-C1M-Cre minigene corrected) compared with uncorrected (P47-04), healthy control iPSCs, or healthy control peripheral blood. The DHR assay, gating for MFI assessment, and donor target assessment are as described in the Figure 2D legend. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Functional analysis of myeloid cells generated from homozygous Δ734-748 NCF1 p47-CGD iPSCs corrected by exon 2 replacement. Functional analysis of myeloid cells generated from homozygous Δ734-748 NCF1 p47-CGD iPSCs corrected by exon 2 replacement. (A) p47phox expression in myeloid cells differentiated from iPSCs derived from the homozygous Δ734-748 NCF1 p47-CGD subject, including the uncorrected line, P47-05 (first panel on the left), 7 exon 2 replacement (GT ins) clones, P47-05-C1 to C7 (middle 7 panels), and the healthy control iPSCs (last panel on the right). (B) Oxidase function in corresponding clones is shown by DHR assay. The DHR assay, gating for MFI assessment, and donor target assessment are as described in the Figure 2D legend. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Schematic diagram of sequencing determined SNP detection at TI sites. Schematic diagram of sequencing determined SNP detection at TI sites. Forward and reverse sequencing in duplicate was performed on the ∼2-kb PCR products amplified from genomic DNA of donor C TI gene-corrected p47-CGD iPSCs. PCR products were generated from 2 primer sets, one amplifying from 5′ of the first distinguishing SNP in intron 1 to the puromycin cassette and the other from the puromycin cassette to 3′ of the last distinguishing SNP in intron 2 of NCF1 or its pseudogenes. The highlighted regions are color-coded based on the SNP’s gene identity with blue as NCF1 (1), orange as NCF1B pseudogene (1B), and yellow as NCF1C pseudogene (1C). Some SNPs represent only NCF1, NCF1B, or NCF1C, while others can represent 2 different potential combinations. The single-color rows for 16 of 18 clones indicate unambiguous concordance across all SNPs in both primer products, allowing single-locus assignment of the TI. Crosshatch rows for 2 clones represent discordance of SNPs across or between the primer products, suggestive of the presence of TI at 2 loci. The first PCR product contained 3 potential SNPs, and the second PCR product contained exon 2 and multiple SNPs. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology

Functional analysis of gene-targeting correction of p47-CGD patient HPCs. HPCs from p47-CGD subject 3 (homozygous exon 2 ΔGT NCF1) were cultured in Stemspan SFEMII supplemented with stem cell factor, Flt3 ligand, and thrombopoietin and gene targeted with ZFN mRNA and donor B or donor D rAAV6 at day 2 of culture and analyzed at day 10 of culture under conditions inducing myeloid differentiation with 50 ng/mL granulocyte colony-stimulating factor. Functional analysis of gene-targeting correction of p47-CGD patient HPCs. HPCs from p47-CGD subject 3 (homozygous exon 2 ΔGT NCF1) were cultured in Stemspan SFEMII supplemented with stem cell factor, Flt3 ligand, and thrombopoietin and gene targeted with ZFN mRNA and donor B or donor D rAAV6 at day 2 of culture and analyzed at day 10 of culture under conditions inducing myeloid differentiation with 50 ng/mL granulocyte colony-stimulating factor. Shown first on the left are uncorrected myeloid differentiating HPCs from the patient in whom no oxidase-positive cells were detected. Shown last on the right are myeloid differentiating HPCs from a healthy control, where 33% of cells fall into the oxidase-positive gate. Shown on the middle-left and on the middle-right panels, respectively, are myeloid differentiating minigene (donor B) or exon 2 replacement (donor D) corrected HPCs from the patient where significant numbers of oxidase-positive cells are detected with MFI approaching that of a healthy control subject. Randall K. Merling et al. Blood Adv 2017;1:270-278 ©2017 by American Society of Hematology