Gene therapy was first proposed as a viable therapeutic strategy for genetic disease nearly 30 years ago.
There are many notable recent successful gene therapy trials for multiple diverse diseases; however, gene
therapy for cystic fibrosis (CF) has presented many unexpected roadblocks. For example, 1) airway cells have
evolved many barriers to prevent uptake of foreign DNA, 2) mouse models of CF generally lack lung disease,
and 3) each of the standard gene delivery vehicles have pros and cons. We have a demonstrated track record
of utilizing many categories of viral and non-viral based reagents for gene delivery to the airways. Viral based
vectors are the most efficient way to deliver genes to airway cells. Our long-term goal is to engineer a reagent
for gene delivery to cells that is an effective therapeutic for CF lung disease, yet there is a critical need for
improved gene delivery tools. Here, we develop a novel reagent. We propose to demonstrate that our
engineered vector can integrate into the genome of progenitor cells in the airways and express the transgene
in the appropriate surface cells. To this end, we have combined the efficiency of an adenoviral-based vector
with the persistent expression of a DNA transposon-based non-viral vector. In addition, we test this `hybrid'
vector in a relevant large animal model of CF. Here we demonstrate that this vector will: 1) deliver CFTR to CF
cells and correct the anion transporter defect in vitro using a novel strategy to focus cellular integration and
expression, 2) verify that the necessary levels of gene transfer are achievable in pigs airways in vivo, and 3)
correct the anion channel defect and disease progression in the airways of a pig CF model. We investigate a
gene delivery tool that can be used to address questions that no other reagent can address. There are >2000
known disease causing mutations in CFTR. Our goal is to provide a life-long gene replacement strategy that
would be efficacious regardless of the disease causing mutation. This proposed research is highly innovative.
Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral delivery
presents an exciting new opportunity to improve the utility of delivery vehicles for therapeutic genes such as
CFTR. The reagents, methods, and data generated by these experiments will provide guidance for gene
therapies for other monogenic disorders, thereby significantly advancing the gene therapy field.