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Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucle...

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

Sustained expression of the CFTR gene is a major challenge to gene therapy with either viral or nonviral vectors with immune response to vector and transgene products. One strategy to achieve sustained CFTR expression is to modulate the host immune system through transient immunosuppression. In this study, we examined cyclophosphamide (cytoxan), dexamethasone (Dex), and a combination of cyclosporin, methylprednisolone, and azathioprine (combination) for their effects on long-term expression of the human CFTR delivered with helper-dependent adenoviral vectors in mouse airways. We found that cyclophosphamide significantly enhanced long-term expression of the transgenic human CFTR and the reporter gene LacZ by reducing host immune responses. Dex administration greatly reduced neutralizing antibody production but had no effect on transgene expression. Treatment with a combination of cyclosporin A, azathioprine, and methylprednisolone affected neither CFTR gene expression nor inflammation. Our data suggest that transient immunosuppression might be a strategy to improve sustained expression in gene therapy.