Restoration of Defective CFTR in Human Nasal Respiratory Epithelial Cells by CFTR Modulators and mRNA Transfection

Singh, Roshani ,Narayan; Horn, Marilia, Marta; Juko, Marin; Kampshoff, Ami; Schmid, Jochen; Omran, Heymut; Zhang, Dandan; Rosenecker, Joseph; Weber, Wolf-Michael; Große-Onnebrink, Jörg,

Zusammenfassung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the atypical ATP-binding cassette (ABC) family that functions as a phosphorylation-regulated epithelial anion channel. Cystic fibrosis (CF) is characterised by variants in the CFTR gene that lead to impaired epithelial chloride–ion transport and increased mucus viscosity. Although CFTR modulators such as Trikafta® have transformed the care of many CF patients, individuals harbouring rare CFTR variants still have no effective treatment options. In this study, we used primary air–liquid interface (ALI) airway cultures obtained from 21 CF patients (pwCF) and 21 healthy controls (HC) to evaluate the therapeutic efficacy of CFTR restoration based on chitosan-mediated CFTR mRNA and modulators. While modulators restored CFTR channel function in most cultures derived from CF patients, those with class I or other rare variants showed no improvement. Chitosan-mediated CFTR mRNA delivery successfully restored CFTR function in ALI cultures of patients carrying rare CFTR variants with limited or no observed clinical response to modulator therapy, assessed by electrophysiology using our newly developed Multi Transepithelial Current Clamp (MTECC) Ussing chamber. This was then confirmed by morphological visualisation of CFTR protein expression in modulator-responsive patient samples using immunofluorescence (IF) staining. IF revealed an increase in CFTR signal and the restoration of epithelial barrier integrity following chitosan-mRNA and modulator treatment as a secondary outcome alongside CFTR functional measurements. Notably, MUC5AC expression, a major gel-forming mucin expressed by airway goblet cells and mucus viscosity were elevated in CF cultures, but were markedly reduced following successful intervention, approaching the levels seen in HCs. These findings establish the potential of chitosan-mRNA delivery as a therapeutic approach for CF patients, particularly those who do not respond to modulators. They also provide a practical, comparative evaluation of advanced mRNA-based treatments in patient-derived airway models.