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A novel lipidic vector composed of DOTAP/Chol liposomes, asialofetuin (AF), protamine sulfate and DNA has been developed. The resulting protamine-AF-lipoplexes improved significantly the levels of gene expression in cultured cells and in the liver upon i.v. administration. Lipoplexes containing the optimal amount of AF (1 mg/mg DNA) showed a 16-fold higher transfection activity in HepG2 cells than nontargeted (plain) complexes. The uptake by cells having asialoglycoprotein receptors (ASGPr) on their plasma membrane was decreased by the addition of free AF, indicating that AF-lipoplexes were taken up specifically by cells via ASGPr-mediated endocytosis. Results from transfections performed in cells defective in ASGPr, ie HeLa cells, confirmed this mechanism. By addition of the condensing peptide, protamine sulfate, smaller complexes were obtained, which enhanced even more the uptake of AFcomplexes in HepG2 cells and in the liver. The optimal amount of protamine was 0.4 mg/mg DNA, and gene expression was about 5-fold over that obtained with AF-lipoplexes in the absence of the peptide, and 75-fold higher than that with plain conventional lipoplexes. Protamine-AF-lipoplexes increased by a factor of 12 luciferase gene expression in the liver of mice administered systemically via the tail vein, compared to plain complexes. In summary, our findings extend the scope of previous studies where AF-lipoplexes were used to introduce DNA into hepatocytes. The combination of targeting and protamine condensation obviated the need for partial hepatectomy, commonly required to obtain efficient gene delivery in this organ. Since protamine sulfate has been proven to be non-toxic in humans, the novel liverspecific vector described here may be useful for the delivery of clinically important genes to this organ.

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Cationic liposomes and the complexes they form with DNA (lipoplexes) constitute the most promising alternative to the use of viral vectors for gene therapy. One of the limitations to their application in vivo, however, is the inhibition of gene delivery by serum. In a previous study, we demonstrated that transferrin (Tf)-lipoplexes were superior to plain lipoplexes in transfecting HeLa cells in the presence of high concentrations of serum. With the goal of obtaining efficient gene expression in vivo, we evaluated the efficacy of Tf-lipoplexes (containing DOTAP and cholesterol) in transfecting primary hepatocytes and adipocytes in the presence of high serum concentrations. The association of transferrin with cationic liposomes increased luciferase expression compared to plain lipoplexes in primary cells as well as in HepG2 and 3T3-L1 differentiated adipocytes. The complexes were not cytotoxic and were highly effective in protecting DNA from attack by DNase I. An efficient and reliable method was developed to prepare lipoplexes containing both Tf and protamine sulfate, where the latter was mixed with transferrin, followed by the addition of cationic liposomes and DNA. The resulting protamine-Tf-lipoplexes increased significantly the levels of gene expression in cultured cells and in various tissues in mice following i.v. administration.