Targeted replacement of full-length CFTR in human airway stem cells by CRISPR-Cas9 for pan-mutation correction in the endogenous locus

Abstract

Cystic fibrosis (CF) is a monogenic disease caused by impaired production and/or function of the CF transmembrane conductance regulator (CFTR) protein. Although we have previously shown correction of the most common pathogenic mutation, there are many other pathogenic mutations throughout the CF gene. An autologous airway stem cell therapy in which the CFTR cDNA is precisely inserted into the CFTR locus may enable the development of a durable cure for almost all CF patients, irrespective of the causal mutation. Here, we use CRISPR-Cas9 and two adeno-associated viruses (AAVs) carrying the two halves of the CFTR cDNA to sequentially insert the full CFTR cDNA along with a truncated CD19 (tCD19) enrichment tag in upper airway basal stem cells (UABCs) and human bronchial epithelial cells (HBECs). The modified cells were enriched to obtain 60%-80% tCD19⁺ UABCs and HBECs from 11 different CF donors with a variety of mutations. Differentiated epithelial monolayers cultured at air-liquid interface showed restored CFTR function that was >70% of the CFTR function in non-CF controls. Thus, our study enables the development of a therapy for almost all CF patients, including patients who cannot be treated using recently approved modulator therapies.

Keywords: CF; CFTR correction; CRISPR-Cas9; airway stem cell therapy; cystic fibrosis; genome editing for CF; universal CFTR correction.

Graphical abstract

Figure 1

Expansion and characterization of UABCs (A and B) UABCs cultured in tissue-cultured plates coated with iMatrix (recombinant laminin 511) showed improved proliferation (A) immediately after plating from tissue and (B) during subsequent passages. Error bars indicate standard deviation. (C) Telomerase activity in the expanded UABCs was measured using telomeric repeat amplification protocol (TRAP), and telomerase activity was observed in UABCs across multiple passages. (D and E) Significant shortening of telomere lengths was also not observed in expanded UABCs when telomere lengths were probed using (D) Southern blot or (E) qPCR.

Figure 2

Insertion of the CFTR cDNA in UABCs and HBECs (A) Schematic of the universal strategy with the two halves of the CFTR cDNA and the tCD19 enrichment cassette. (B) After editing, 2%–4% of edited UABCs were tCD19⁺. Edited UABCs were enriched by FACS to obtain >60% tCD19⁺ cells. A log base 2 scale has been used to make it easier to visualize the enrichment.Error bars indicate standard deviation. (C) UABCs and HBECs from CF donors were edited using the universal strategy. In this representative sample, 83% of the edited UABCs were positive for both tCD19 and KRT5. Controls treated with AAV but without Cas9 were KRT5⁺ and were negative for tCD19. (D) UABCs and HBECs from donors with different genotypes were corrected using the universal strategy. In most cases, >60% of enriched UABCs were tCD19⁺KRT5⁺. Error bars indicate standard deviation. (E–H) >90% of FACS-enriched UABCs were also positive for (E) p63, (F) KRT14, (G) ITGA6, and (H) NGFR.

Figure 3

Restoration of CFTR expression and function in differentiated epithelial sheets (A) Epithelial sheets generated by differentiation of UABCs (cultured on iMatrix) in air-liquid interfaces (ALIs) contained cells positive for KRT5, acetylated tubulin, and mucin 5AC. Scale bar corresponds to 10 μm. (B) Epithelial sheets generated from UABCs cultured on iMatrix (unedited and universal edited) showed trans-epithelial resistances that were not significantly different from epithelial sheets generated from UABCs cultured on BME. (C) The percent alleles modified was quantified both right after sorting and after differentiation for 28−35 days on ALI cultures. The percent of tCD19⁺ alleles was not significantly different between these time points, indicating that the modified cells did not have any disadvantage in proliferation when compared to the unmodified tCD19⁻ cells. (D) Western blot probing CFTR expression. CFTR expression in Calu-3 cells was used as a positive control (lane 1). Samples were diluted by 1/10 relative to other samples since Calu-3 cells are known to express high levels of CFTR. Lane 2 shows strong mature CFTR expression (band C) in a corrected CF sample, which is comparable to the mature CFTR expression (band C) observed in a non-CF control shown in lane 4. Uncorrected cells (F508del/3659delC) obtained from the same donor as the corrected cells used in lane 2 show reduced mature CFTR expression (band C, lane 3). (E) Representative traces obtained from epithelial sheets by Ussing chamber analysis in one non-CF control sample and one CF sample from donor 1 before and after correction. The traces from the corrected sample show similar forskolin and CFTR_inh-172 responses as the non-CF control sample. (F) CFTR_inh-172 short-circuit currents observed in corrected CF samples from 11 different donors as a percentage of the average CFTR_inh-172 currents observed in non-CF controls. (G) CFTR_inh-172 short-circuit currents observed in non-CF controls and epithelial monolayers derived from CF UABCs before and after correction. (H) CFTR_inh-172 short-circuit currents observed in non-CF controls and epithelial monolayers derived from CF HBECs before and after correction.