Author: Stecenko Arlene King Gayle Torii Keizo Gao Xiang Persmark Magnus Shih Kent Brigham Kenneth Raja-Walia Rampyari
Publisher: Informa Healthcare
ISSN: 1521-0499
Source: Experimental Lung Research, Vol.26, Iss.3, 2000-04, pp. : 179-201
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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.
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