title: Enhancement of liposome-mediated gene transfer to human airway epithelial cells by replication-deficient adenovirus.
abstract: We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-mediated gene transfer in cultured human airway epithelial cells. Pl/L/Ad complexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a high-molecular-weight, low-mobility complex, the diameter of which was 300 to 350 nm. Compared to pl/L alone, pl/L/Ad enhanced luciferase expression on average by 1 log-fold in human airway epithelial cells which express either mutant or wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Transgene expression was sustained at high levels for up to 7 days following transfection with pl/L/Ad. Using a heat-stable alkaline phosphatase reporter gene, we showed that a larger fraction of cells was transfected by pl/L/Ad compared to pl/L. Finally, cells were exposed to Ad for 0 to 24 hours prior to pl/L or exposed to pl/L prior to Ad. We found that enhancement was significantly greater using pl/L/Ad compared to the simultaneous addition of Ad with the pl/L complexes. In addition, when pl/L was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid DNA remained in a compartment in the cell for several hours and became available for transcription with the addition of Ad. When Ad was added prior to pl/L, enhancement was found suggesting that the effect of the virus on cell membranes may persist for up to 24 hours. We conclude that the tripartite pl/L/Ad complex significantly enhances liposome-mediated transgene expression for a prolonged period of time in human bronchial epithelial cells.
authors.affiliation: Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2650, USA.
authors.forename: A
authors.initials: A
authors.lastname: Stecenko
authors.forename: G
authors.initials: G
authors.lastname: King
authors.forename: K
authors.initials: K
authors.lastname: Torii
authors.forename: X
authors.initials: X
authors.lastname: Gao
authors.forename: M
authors.initials: M
authors.lastname: Persmark
authors.forename: K
authors.initials: K
authors.lastname: Shih
authors.forename: K
authors.initials: K
authors.lastname: Brigham
authors.forename: R
authors.initials: R
authors.lastname: Raja-Walia
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D002470:Cell Survival; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis; D018014:Gene Transfer Techniques; D006801:Humans; D008081:Liposomes / Q000235:genetics / Q000378:metabolism; D008722:Methods; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000166:cytology*; D013997:Time Factors; D014162:Transfection
chemical_list: C505032:CFTR protein, human; D004274:DNA, Recombinant; D008081:Liposomes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator