Widespread airway distribution and short-term phenotypic correction of cystic fibrosis pigs following aerosol delivery of piggyBac/adenovirus

Abstract

Cystic fibrosis (CF) is a common genetic disease caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Although CF affects multiple organ systems, chronic bacterial infections and inflammation in the lung are the leading causes of morbidity and mortality in people with CF. Complementation with a functional CFTR gene repairs this defect, regardless of the disease-causing mutation. In this study, we used a gene delivery system termed piggyBac/adenovirus (Ad), which combines the delivery efficiency of an adenoviral-based vector with the persistent expression of a DNA transposon-based vector. We aerosolized piggyBac/Ad to the airways of pigs and observed widespread pulmonary distribution of vector. We quantified the regional distribution in the airways and observed transduction of large and small airway epithelial cells of non-CF pigs, with ∼30–50% of surface epithelial cells positive for GFP. We transduced multiple cell types including ciliated, non-ciliated, basal, and submucosal gland cells. In addition, we phenotypically corrected CF pigs following delivery of piggyBac/Ad expressing CFTR as measured by anion channel activity, airway surface liquid pH, and bacterial killing ability. Combining an integrating DNA transposon with adenoviral vector delivery is an efficient method for achieving functional CFTR correction from a single vector administration.

Engineered amphiphilic peptides enable delivery of proteins and CRISPR-associated nucleases to airway epithelia

“…This is because the respiratory epithelium presents barriers through its specialized cell types, secreted host defense factors, and mucociliary transport 1 . While in vivo delivery with vector systems has advanced [2][3][4][5][6][7] , airway epithelia remain poorly transduced by many viral and non-viral approaches [8][9][10][11][12] .…”

Extracellular Vesicle-Mediated siRNA Delivery, Protein Delivery, and CFTR Complementation in Well-Differentiated Human Airway Epithelial Cells

“…CFTR complementation restores the anion channel defect in well-differentiated human airway epithelial cells derived from CF donors (HAE CF ) [43,44]. Typically, viral vectors are required to deliver the CFTR cDNA.…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…Yant et al [147] showed for the first time that systemic delivery of HDAd vectors carrying a SB system with a human factor IX transgene resulted in integration and persistent factor IX expression in mice. More studies have reported gene transfer by Ad vectors expressing other transposon systems, such as PB [152], T2TP [153], and bacteriophagederived integrase PhiC31 [154]. Through molecular evolution, a new generation of hyperactive SB transposase (SB1009) exerted 100-fold enhancement in efficiency and strong activity in human CD34 + cells without affecting their multipotency [148].…”

“…We have demonstrated the efficacy of SB1009mediated integration into HSCs by HDAd5/35++ vectors in several disease models, including b-hemoglobinopathies, hemophilia A, and Fanconi anemia (FA) [150,151]. More studies have reported gene transfer by Ad vectors expressing other transposon systems, such as PB [152], T2TP [153], and bacteriophagederived integrase PhiC31 [154]. Delivery of these transposons/integrases to HSCs by HDAd vectors remains to be fully explored.…”