dioleoyl-phosphatidylethanolamine and Cystic-Fibrosis

Cationic lipids facilitate plasmid delivery, and some cationic sterol-based compounds have antimicrobial activity because of their amphiphilic character. These dual functions are relevant in the context of local ongoing infection during intrapulmonary gene transfer for cystic fibrosis. The transfection activities of two cationic lipids, dexamethasone spermine (DS) and disubstituted spermine (D(2)S), were tested as individual components and mixtures in bovine aortic endothelial cells and A549 cells. The results showed a 3- to 7-fold improvement in transgene expression for mixtures of DS with 20 to 40 mol% D(2)S. D(2)S and coformulations with DS, dioleoyl phosphatidylethanolamine, and DNA exhibited potent bactericidal activity against Escherichia coli MG1655, Bacillus subtilis, and Pseudomonas aeruginosa PAO1, which was maintained in bronchoalveolar lavage fluid. Complete bacterial killing was demonstrated at approximately 5 microM, including gene delivery formulations, with 2 orders of magnitude higher tolerance before eukaryotic membrane disruption (erythrocyte hemolysis). D(2)S also exhibited lipopolysaccharide (LPS) scavenging activity resulting in significant inhibition of LPS-mediated activation of human neutrophils with 85 and 65% lower interleukin-8 released at 12 and 24 h, respectively. Mixtures of DS and D(2)S can improve transfection activity over common lipofection reagents, and D(2)S has strong antimicrobial action suited for the suppression of bacterial-mediated inflammation.

Topics: Animals; Anti-Infective Agents; Cations; Cattle; Cystic Fibrosis; Dexamethasone; DNA; Eukaryota; Gene Transfer Techniques; Genetic Therapy; Humans; Interleukin-8; Lipids; Phosphatidylethanolamines; Plasmids; Spermine; Transfection; Transgenes

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Topics: Analysis of Variance; Animals; Autoradiography; Bronchi; Cations; Chloramphenicol O-Acetyltransferase; Cystic Fibrosis; Epithelial Cells; Gene Expression; Gene Transfer Techniques; Genetic Therapy; Immunohistochemistry; Injections, Intravenous; Liposomes; Mice; Mice, Inbred Strains; Phosphatidylethanolamines; Pulmonary Alveoli; Quaternary Ammonium Compounds; Trachea

Complexes of DNA and cationic lipids are promising vectors for gene transfer. Most cationic lipid formulations contain both a cationic component and a neutral co-lipid. We found that the co-lipid could influence DNA uptake in COS-1 cells, but processes subsequent to uptake were even more important in determining gene expression. We compared dioleoylphosphatidylethanolamine (DOPE) and structural analogs of DOPE combined with cationic lipids and found that DNA uptake and transgene expression did not always correlate. Transgene expression was dependent on DNA uptake into the cell, on entry of DNA into the cytoplasm, and on release of DNA from the lipid complex. We found that some co-lipids had a greater effect on DNA uptake, whereas others had a greater effect on steps subsequent to entry. Based on those results, we tested the hypothesis that co-lipids conferring different properties could be combined to enhance gene transfer. The results showed that a combination of co-lipids had a synergistic effect on expression. We also found that structural analogs of DOPE were more effective than DOPE in enhancing gene transfer to mature human airway epithelia studied in vitro and to mouse lung studied in vivo. These data provide insight into the mechanism by which co-lipids influence cationic lipid-mediated gene transfer and show that optimization of the effects of co-lipids can enhance gene transfer both in vitro and in vivo.

Topics: Animals; Cations; COS Cells; Cystic Fibrosis; Epithelium; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Humans; Lipids; Lung; Mice; Phosphatidylethanolamines; Transgenes