Functional Restoration of gp91phox-Oxidase Activity by BAC Transgenesis and Gene Targeting in X-linked Chronic Granulomatous Disease iPSCs

Abstract

Chronic granulomatous disease (CGD) is an inherited immunodeficiency, caused by the inability of neutrophils to produce functional NADPH oxidase required for fighting microbial infections. The X-linked form of CGD (X-CGD), which is due to mutations in the CYBB (gp91phox) gene, a component of NADPH oxidase, accounts for about two-thirds of CGD cases. We derived induced pluripotent stem cells (iPSCs) from X-CGD patient keratinocytes using a Flp recombinase excisable lentiviral reprogramming vector. For restoring gp91phox function, we applied two strategies: transposon-mediated bacterial artificial chromosome (BAC) transgenesis and gene targeting using vectors with a fixed 5' homology arm (HA) of 8 kb and 3'HA varying in size from 30 to 80 kb. High efficiency of homologous recombination (up to 22%) was observed with increased size of the 3'HA. Both, BAC transgenesis and gene targeting resulted in functional restoration of the gp91phox measured by an oxidase activity assay in X-CGD iPSCs differentiated into the myeloid lineage. In conclusion, we delivered an important milestone towards the use of genetically corrected autologous cells for the treatment of X-CGD and monogenic diseases in general.

Figure 1

Derivation of X-linked chronic granulomatous disease (X-CGD) induced pluripotent stem cells (iPSCs) from plucked hair keratinocytes. (a) Diagram of the lentiviral reprogramming vector (LV-OKSM-Tomato). (b) Representative images of hair outgrowth, keratinocytes, and a derived iPSC colony (bars: left panel = 200 µm; middle and right panels = 100 µm). (c) Southern blot hybridized with the probe indicated in a showing lentiviral integrations in X-CGD iPSCs clones. Clone S22 carries three integrations and clone J14 two integrations. (d) The X-CGD iPSCs express endogenous pluripotency genes in similar levels to hESC line H7.S6. Data represent mean and SD of samples collected at day 100 and at day 125 and analyzed in triplicates. (e) X-CGD iPSCs express the pluripotency markers OCT4, NANOG, SSEA-3, SSEA-4, TRA-1–60, and TRA-1–81 and are negative for SSEA-1. Counterstaining with Hoechst 33342 and overlay is shown (bar = 100 µm). (f) X-CGD iPSCs gave rise to all three germ layers in teratoma formation assays. hESC, human embryonic stem cell.

Figure 2

Transposition-mediated bacterial artificial chromosome (BAC) transgenesis. (a) Schematic representation of the 175-kb BAC containing the CYBB gene. The CYBB exons are shown as black rectangles. The bacterial backbone contains the chloramphenicol resistance gene (CmR) and the origin of replication (ori). The ubiquitin-C-BSD-pA selection cassette together with the PB3 inverted terminal repeat (ITR) was inserted on one side of the bacterial backbone. On the other side, a cassette was inserted containing the herpes simplex virus thymidine kinase negative selection marker (HSV-TK) together with an ampicillin resistance gene (ampR) and the PB5 ITR. The modified CYBB BAC was co-lipofected with the hyPBase transposase expression vector. Upon transposition, the BAC integrates as a full-length copy from the PB3 to PB5 ITR excluding the bacterial backbone. Inverted arrows indicate the PCRs for detecting transposition signature. Splinkerette PCR was used to identify the genomic integration site. (b) Table showing transposition events in H7.S6 human embryonic stem cells (hESCs) and X-linked chronic granulomatous disease (X-CGD) induced pluripotent stem cell (iPSC) clones S22 and J14. (c) Mapping of the BAC integration sites in the human genome. Red triangles mark the integration site into the human chromosomes. (d) BACs integrated into TTAA sites, a characteristic of piggyBac transposition. (e) Mapping of the BAC integration sites using fluorescence in situ hybridization for clone 5. DNA of the CYBB BAC was directly labeled in red and hybridized together with a control probe for the centromere of chromosome X (where the endogenous CYBB gene is located) labeled in green. A representative metaphase is shown with a red and a green signal on the X chromosome (endogenous CYBB gene and centromere region of chromosome X) and an additional red signal on one chromosome 2 that corresponds to the integration of the CYBB BAC (arrow).

Figure 3

Correction of the X-linked chronic granulomatous disease (X-CGD)–causing mutation by gene targeting. (a) Schematic representation of the targeting strategy and Flp recombination. In the targeting construct, a PGK-neo-pA selection cassette flanked by FRT sites was inserted in intron 8 of the CYBB gene close to the mutation in exon 9 (asterisks) in a bacterial artificial chromosome (BAC) vector. The BAC was trimmed upstream of the selection cassette to get a 5′HA of 8 kb. To obtain variable sizes of the 3′HA, the BAC was digested with SalI (32-kb 3′ homology arm (HA)), SbfI (54-kb 3′HA), or SfiI (80-kb 3′HA). (b) Southern blot of genomic DNA from G418 resistant clones digested with ApaLI and hybridized with an external 5′ probe shown in a. Homologous recombination result in a 11.6-kb band (TG), whereas the endogenous unrecombined is 10 kb. Asterisks indicate the targeted clones. (c) Southern blot analysis of X-CGD–targeted clones after Flp recombination. Upper panel: excision of the PGK-neo cassette results in a band (Δneo^Flp) that runs at the same size as the endogenous unmodified allele. Middle panel: Flp recombination resulted in the simultaneous excision of the reprogramming lentiviral vector in all clones that lost the PGK-neo cassette. Arrows point at the three lentiviral integrations. Lower panel: as a loading control, the same blot in middle panel was hybridized with the 5′ external probe. (d) Sequencing results confirming the correction of the mutation in six targeted clones. (e) Conventional cytogenetics (G-banding) of one correctly targeted and Flp-recombined clone revealed a normal male karyotype (46,XY).

Figure 4

Functional restoration of oxidase activity in X-linked chronic granulomatous disease (X-CGD) induced pluripotent stem cells (iPSCs) differentiated into the hematopoietic lineage. Representative analyses are shown. (a) Diagram of the differentiation protocol. (b) Flow cytometry analysis of the hematopoietic markers CD34 and CD45 at differentiation days 18–20. (c) Flow cytometry analysis of the myeloid marker CD13 at differentiation days 43–44. (d) Expression of gp91phox at differentiation days 43–44. Note that the X-CGD granulocytes do not express gp91phox, whereas the differentiated iPSCs containing the CYBB bacterial artificial chromosome (BAC) express high levels. Differentiated human hematopoietic stem and progenitor cells (HSPCs) and granulocytes from healthy individuals serve as positive control. (e) X-CGD granulocytes do not show any oxidase activity measured by a chemiluminescence assay. Granulocytes from unaffected individuals produced superoxide in a cell-number–dependent manner. (f) Oxidase activity was undetectable in differentiated X-CGD iPSCs but was restored in the ones carrying the BAC transgene. In vitro differentiated HSPCs serve as positive control. (g) Restoration of the oxidase activity was also observed with the correctly targeted and Flp-recombined differentiated X-CGD iPSCs.