Efficiency of plasmid delivery and expression after lipid-mediated gene transfer to human cells in vitro

Z Bebok, AM Abai, JY Dong, SA King, KL Kirk, G Berta, BW Hughes, AS Kraft, SW Burgess, W Shaw, PL Felgner and EJ Sorscher

Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, USA.

Cationic liposome-mediated gene transfer has become increasingly important in the development of experimental therapies for human diseases, such as melanoma, human immunodeficiency virus infection, cystic fibrosis and alpha-1 antitrypsin deficiency. However, very little is known about the mechanisms by which lipid-mediated gene transfer occurs. We studied the kinetics of plasmid delivery and expression by using this technique. Plasmid entry in the cystic fibrosis respiratory epithelial cell line 2CFSME0-1 as well as in two other cell lines (HeP 2g and HeLa) occurred in 95 to 100% of cells within 1 hr of the initiation of lipid-mediated gene transfer. In hepatic and respiratory cells, transcription of a construct containing the cystic fibrosis transmembrane conductance regulator was observed in more than 80% of the cell population; similarly high levels of plasmid utilization were obtained in studies of HLA-B7 expression in human melanoma cells. Studies directly relevant to current human trials of lipid-mediated gene transfer indicate that plasmid entry, transcription and translation are often surprisingly efficient, and may occur in nearly 100% of human cells in culture when sensitive methods for detection are used. Furthermore, conventional X-gal immunohistochemistry markedly underestimates transfection efficiency during transient gene expression. These studies point to a new mechanistic understanding of the features that limit expression by using cationic liposomes.

Volume 279, Issue 3, pp. 1462-1469, 12/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

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