Résumé :
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Duchenne muscular dystrophy (DMD) is one of the most severe myopathies caused by mutations in the dystrophin gene. Gene therapy is being investigated to introduce by non viral vectors, the normal dystrophin gene into skeletal muscle cells. The knowledge of the uptake by the cells of these vectors and the intracellular trafficking of the plasmid DNA (pDNA) are of crucial importance to design competitive nonviral gene delivery systems. We have compared the uptake and intracellular routing in the mouse myoblast cells C1C12 line, of pDNA complexed either with a cationic polymer (His-lPEI-polyplexes), cationic liposomes (Lip100 lipoplexes) or polymer/liposomes (LPD100 lipopolyplexes). The peculiarity of these vectors is that they contain histidine or imidazole groups that give them the ability to destabilize the endosome to facilitate the passage pDNA into the cytosol. With the luciferase gene, we found that His-lPEI polyplexes gave the highest luciferase expression which was three and ten fold higher than with LPD100 lipopolyplexes and Lip100 lipoplexes, respectively. C2C12 cells showed cell viability above 78% when the transfection was carried out with His-lPEI polyplexes and LPD100 lipopolyplexes and 68% with Lip100 lipoplexes. To identify the uptake process of these complexes, we developed C2C12 clones stabilized with chimeric EGFP-protein, specific of intracellular compartments involved in endocytosis pathway. Real time confocal laser scanning microscopy was performed with C2C12 cells expressing either fluorescent early endosomes (Rab5-EGPP), late endosomes (Rab7-EGFP) or caveolae (Cav1-EGFP), respectively. We demonstrated by colocalization experiments with a fluorescent-labelled pDNA that His-lPEI polyplexes are preferentially internalized by clathrin-mediated endocytosis, while LPD100 lipopolyplexes and Lip100 lipoplexes are taken up by caveolae-mediated endocytosis. Acknowledgements: This work is supported by Association Franse contre les Myopathies (AFM) within a French consortium.
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