Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells

Abstract

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Figure 1

ZFN-Mediated Correction of ΔI507 or ΔF508 CFTR Mutations in CF iPSCs (A) Outline of methodology involving co-delivery of CFTR-specific ZFNs together with CFTR donor, followed by Cre-recombinase-mediated excision. (B) The schematic shows the expected genomic organization of a targeted CFTR allele including the WT exon 10 (shown in black) together with the pgk-puroTK selection cassette. A unique 6.4-kb hybridizing band is expected for a correctly modified clone and is apparent in the four corrected clones (17-14, 17-1, 17-16, and 17-9), but absent in the Cre-excised clones and the non-targeted clone 17 CF iPSCs. (C) Sequence chromatograms of the modified WT and unmodified ΔF508 CFTR alleles from corrected CF iPSC clones. See also Figure S1.

Figure 2

Cre-Mediated Excision of puroTK Cassette from Corrected CF WT/ΔF508 iPSCs (A) Schematic of the modified allele, before and after Cre-mediated excision, and the unmodified allele. The location of PCR primers (arrows marked 3 and 3′), both located outside of donor sequences, used in verification by amplification are shown. Also indicated are the expected sizes of Cla I digestion products. (B) (Top) The PCR amplicons for the original targeted clones (17-9 and -16), the Cre-excised clones (17-9-C1 and -C2; 17-16-C1 and -C2), and the original clone 17 CF iPSCs. The presence of only the 1.8-kb band for Cre-excised clones is consistent with successful excision. (Bottom) The results of Cla I digestion of the PCR amplicons. The size of bands for the Cre-excised clones is consistent with successful excision. (C) RT-PCR analysis of CFTR expression for two targeted CF iPSCs (17-9 and -16) as well as their derived Cre-excised clones. Also shown is CFTR expression by the original clone 17 CF iPSCs and CFTR-expressing Calu-3 cells; mouse embryo fibroblasts (MEFs) and HEK293TN cells are negative controls. (D) Sequencing of CFTR RT-PCR product from mutant ΔI507/ΔF508 CFTR iPSCs (clone 17) revealed a mixture of ΔI507 and ΔF508 CFTR sequences. (E) Sequencing of CFTR RT-PCR product from corrected WT/ΔF508 CFTR iPSCs (clones 17-9 and 17-16), together with their Cre-excised derivatives, revealed a mixture of WT and ΔF508 CFTR sequences. See also Figure S2 and Tables S1 and S2.

Figure 3

In Vitro Differentiation of Corrected CF WT/ΔF508 iPSCs (A) Outline of the defined, step-wise differentiation protocol used to generate anterior foregut cell fates. (B) Gene expression analysis of day 19 differentiated mutant (17 and 28), corrected CF iPSCs (17-9-C1, 17-14-C1, and 17-16-C1), and WA09 hESCs indicates upregulation of lung and pan-endodermal markers. Data (mean ± SD, three well replicates) from a representative experiment further characterized in Figures 3C and 3G. See also Figure S3E. (C) Western blot analysis of protein lysates from day 19 differentiation cultures probed with a CFTR-specific antibody. Detection of Calnexin demonstrated equal sample loading for differentiated iPSC/hESC samples. See also Figures S3D and S3F. (D) Representative short-circuit current (Isc) traces of epithelial monolayers differentiated from mutant (17) and corrected (17-16-C1) CF iPSC by Ussing chamber analysis. Cells were treated with either DMSO (0.03%) or VX809 (3 μM) for 24 hr. After establishment of Cl⁻ gradient and the addition of amiloride, monolayers were treated with forskolin and genistein followed by the administration of CFTR inhibitor 172. The change in Isc (μA/cm²) for each perturbation is shown. Only corrected clone 17-16-C1 demonstrates the presence of CFTR channels in the cell membrane, as evidenced by activation of cAMP-dependent short-circuit currents. The addition of CF corrector VX809 to differentiated clone 17 sample partially restores CFTR-mediated chloride activity. (E) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17), with or without CF corrector VX809, and corrected (17-16-C1) CF iPSC (three transwell replicates per sample, mean ± SD. (F) Aggregated data of short-circuit current measurements from an experiment with differentiated mutant (17) and corrected (17-16-C1), with or without CF corrector VX809, and WA09 hESC control (five transwell replicates per sample, mean ± SD). (G) Aggregated data of short-circuit current measurements from an experiment including all independent mutant (17 and 28) and corrected (17-9-C1, 17-14-C1, and 17-16-C1) differentiated CF iPSC clones (three to six transwell replicates per sample, mean ± SD). (H) Aggregated data of short-circuit current measurements; graphed is the maximum change in short-circuit current resulting from the addition of forskolin and genistein. Shown is the mean ± SE. The comparison shown is between two mutant CF clones (17 and 28; total of four independent differentiated experimental samples, three to six transwell replicates per sample) and three corrected CF clones (17-9-C1, 17-14-C1, and 17-16-C1; total of five independent differentiated experimental samples, three to six transwell replicates per sample). Results were clustered by clonal cell line and experiment number; statistical analysis was performed using a linear mixed-effect model by restricted maximum likelihood to account for correlated replicates within the same experiment. See also Figure S3.

Figure 4

Allele-Preferred Targeting of CF iPSCs (A) Schematic of donor 1 and donor 2 engineered with respective A or G base change at intron 9; results from a total of two independent targeted-integration experiments with either donor 1 or donor 2 are shown. (B) Schematic of uncorrected CFTR alleles ΔI507 and ΔF508, highlighting A or G base within intron 9.