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.