C19-04 Autologous in Vivo Basal Cell Therapy Shows Reconstitution of Airway Epithelium in Wild Type Ferrets

Rationale Mutation-agnostic genetic therapies are urgently needed for cystic fibrosis (CF). Airway basal cells are ideal for CFTR editing because of their self-renewal and multi-lineage potential. Ferrets provide a relevant model due to their airway similarity to humans and recapitulation of CF physiology. Here, we established preclinical proof of concept using an ex vivo ferret model to test engraftment of ferret bronchial epithelial cells (FBECs) on injured trachea, then performed in vivo autologous FBEC transplantation in wild-type (WT) ferrets to assess feasibility of cell-based therapy, including colocalized epithelial function determination. Methods WT FBECs were obtained via bronchoscopy, cultured, and GFP-labeled through lentiviral transduction. For ex vivo studies, excised whole WT tracheae were split along the ventral surface, affixed to a gelatin matrix, and cultured in antimicrobial media following epithelial injury. GFP+ cells were seeded, and tissues were evaluated using histology and Ussing chamber analysis. For in vivo experiments, WT ferrets underwent bronchial brushing to isolate FBECs, which were expanded (>600 million cells), and confirmed to be > 80% GFP+ and 100% basal cells by flow cytometry prior to transplant. Six hours after airway injury with 2% polidocanol GFP+ FBECs were reintroduced into donor ferrets via bronchoscopic instillation. Results Three weeks post-transplant, ex vivo trachea confirmed durable GFP+ engraftment with ciliated, basal, and secretory cell differentiation by immunofluorescence (IF, n = 4). Ussing analysis showed CFTR-dependent functional recovery in GFP+ engrafted tissues compared with injury-only controls. Short-circuit current (Isc) responses to forskolin (ΔIsc: untreated 1060 ± 315.4; injury only 138.5 ± 116.7; injury + GFP+ 361 ± 57.5) and Inh/GlyH inhibition (−979 ± 496.4; −205 ± 237.6; −788.5 ± 137.1 respectively) indicated augmented CFTR activity approaching untreated controls. In in vivo experiments, WT ferrets (n = 2) tolerated autologous FBEC transplantation and remained healthy up to 21 days. Fluorescent microscopy of the transplanted trachea showed successful GFP+ cell engraftment and intact epithelium in > 70% of the trachea at 21 days. IF analysis of the explanted airways confirmed transplanted cells largely exhibited KRT5+ basal cell identity with early evidence of epithelial differentiation at day 7. Conclusion We established proof-of-concept for autologous FBEC transplantation in a ferret model, demonstrating successful engraftment, epithelial regeneration, and partial restoration of CFTR function. Functional ex vivo ferret trachea culture enables evaluation of cell-based airway repair and CF correction strategies. These findings support the feasibility of basal cell-based therapies for CF and provide a foundation for future studies using genetically corrected CF ferret airway cells. This abstract is funded by: CFF