Enhancement of liposome-mediated gene transfer to human airway epithelial cells by replication-deficient adenovirus

We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-me diated gene transfer in cultured human airway epithelial cells. Pl/L/Ad com plexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a h igh-molecular-weight, low-mobility complex, the diameter of which was 300 t o 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 eithe r mutant or wild-type cystic fibrosis transmembrane conductance regulator ( CFTR). Transgene expression was sustained at high levels for up to 7 days f ollowing transfection with pl/L/Ad. Using a heat-stable alkaline phosphatas e 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 pill or exposed to Pl/L prior to Ad. We found that enhanceme nt was significantly greater using pl/L/Ad compared to the simultaneous add ition of Ad with the pill complexes. In addition, when pill was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid D NA remained in a compartment in the cell for several hours and became avail able 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 expressi on for a prolonged period of time in human bronchial epithelial cells.