Poster: 229Helper-dependent adenoviral vector transduces ionocytes in cystic fibrosis nasal epithelium

M. Hao; H. Chen; S. Park; Z. Chen; R. Duan; Z. Zhou; H. Dong; X. Zhang; Y. Tang; J. Qiu; J. Hu; J. Engelhardt; F. Yuan; Z. Yan

doi:10.1016/j.jcf.2026.07.260

Cystic fibrosis (CF) is caused by mutations in the CFTR gene. CF lung disease remains the leading cause of mortality. Effective gene therapy for CF lung disease requires restoration of normal airway homeostasis, which is challenging for the CFTR-addition approach given the marked heterogeneity in CFTR expression across epithelial cell types. To enable investigation of cell-type-specific correction, we developed a new CF ferret model, CFTRint1-eGFP(lsl), in which CFTR is knocked out (KO) by inserting an exon-trap minigene “Lox-eGFP-STOP-Lox” into intron 1, enabling Cre-mediated reactivation. When cultured at at an air-liquid interface (ALI), polarized ferret airway epithelium (FAE) recapitulates pseudostratified airway epithelium containing multiple epithelial cell types, including ciliated cells, secretory cells, basal cells, and ionocytes. Notably, ionocytes are the epithelial cell type that expresses the highest level of CFTR in the proximal airways and are relatively abundant in polarized FAE-ALI derived from nasal basal cells. In this study, nasal FAE-ALI were used to evaluate helper-dependent adenovirus (HDAd) vectors for CFTR repair and replacement. FAE-ALI from CFTRint1-eGFP(lsl)/G551D CF background were apically transduced with HDAd-Cre or HDAd-CFTR, followed by Ussing Chamber assays to assess CFTR function by measuring transepithelial short-circuit current (Isc). To profile the transduced cell types, nasal FAE-ALI cultures derived from Cre-responsive ROSA-TG reporter ferrets were used. Cre-mediated excision of the “Lox-dTomato-STOP-Lox” element resulted in a fluorescence switch from tdTomato to eGFP. Transduced cell types were identified by immunofluorescence staining. Quantitative Ussing chamber measurements showed restoration of CFTR-specific Cl- channel function in the CFTRint1-eGFP(lsl)/G551D CF ALI cultures apically transduced with HDAd-Cre or HDAd-CFTR, but not with control eGFP vectors, indicating successful CFTR repair and replacement. Transient EGTA treatment prior to transduction increased cAMP agonist-stimulated Isc approximately twofold compared to untreated controls. Immunostaining of ROSA-TG ALI cultures demonstrated that HDAd primarily transduced ciliated cells, frequently transduced ionocytes, and less frequently transduced basal cells and secretory cells. The CFTRint1-eGFP(lsl) reporter ferret model represents a valuable system for dissecting CFTR biology and identifying therapeutically relevant cellular targets for CF gene therapy. Given the minimal CFTR expression in ciliated cells, limited vector transduction of secretory cells, and the quiescent state of basal cells in well-differentiated ALI cultures, restored CFTR function likely arises predominantly from edited ionocytes. Consistent with the preferential transduction of ionocytes, efficient CFTR reactivation via Cre-mediated excision of the LSL cassette in the CFTR KO allele supports the conclusion that HDAd effectively delivers gene-editing components to CFTR-expressing cells in the nasal airway epithelium. In addition, HDAd-mediated CFTR expression provides functional compensation through ectopic expression in transduced cells, including ciliated cells. Together, these findings suggest that HDAd-mediated gene delivery enables robust functional rescue of Cl- secretion in the CF airways via CFTR repair and replacement.

Poster: 229Helper-dependent adenoviral vector transduces ionocytes in cystic fibrosis nasal epithelium

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