CELLULAR AND MOLECULAR BARRIERS TO GENE-TRANSFER BY A CATIONIC LIPID

Cationic lipids are widely used for gene transfer in vitro and show pr omise as a vector for in vivo gene therapy applications. However, ther e is limited understanding of the cellular and molecular mechanisms in volved, We investigated the individual steps in cationic lipid-mediate d gene transfer to cultured cell lines, We used DMRIE/DOPE (a 1:1 mixt ure of xy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMR IE) and dioleoyl phosphatidylethanolamine (DOPE)) as a model lipid bec ause of its efficacy and because it is being used for clinical trials in humans, The data show that cationic lipid mediated gene transfer is an inefficient process, Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous , even under conditions that have been optimized to produce the best t ransfection, Inefficiency was not due to inability of the complex to e nter the cells because most cells took up the DNA, However, in contras t to previous speculation, the results indicate that endocytosis was t he major mechanism of entry, After endocytosis, the lipid-DNA aggregat ed into large perinuclear complexes, which often showed a highly order ed tubular structure, Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in t he cytoplasm of most cells, That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer, Finally, b efore transcription can occur, the data show that lipid and DNA must d issociate, These results provide new insights into the physical limita tions to cationic lipid-mediated gene transfer and suggest that attent ion to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applica tions.