Interactions moléculaires et cellulaires entre les protéines E3-14.7K, FIP-1 et les microtubules : application dans le transfert de gènes

Abstract : Gene therapy aims to cure genetic deficiencies and a large variety of acquired diseases by the introduction of nucleic acids into mammalian cells. As an alternative to viral gene delivery vectors, chemical vectors have been developed with reduced immunogenicity, in addition to low cost and ease of production. So far, cationic liposomes and cationic polymers are the most studied and used chemical vectors to transfer therapeutic plasmid DNA (pDNA). Among multiple biological barriers, the limited cytosolic diffusion of pDNA is critical for level of transgene expression. The goal of this work was to identify a dynein-binding peptide which would facilitate the transport of pDNA to the nucleus along microtubules. To this end, we have investigated interaction network of adenoviral protein E3 14.7K. E314.7K is indirectly interacting with Dynein Light Chain TCTEL1 through FIP-1. Different techniques have been used to analyze the protein interactions such as Bioluminescence Resonance Energy Transfer (BRET), Förster Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging (FLIM), immunoprecipitation and Laser Scanning Confocal Microscopy (LSCM). BRET technique allow us to identify a 20 amino acids peptide of E3 14.7K (P79-98) responsible for its interaction with FIP-1. Associated with Quantum Dots P79-98 (P79-98-Qdot) gave colocalizations with isolated microtubules and microtubules in HeLa cells. We have developed the grafting of the peptide directly on the pDNA. Once grafted with P79-98 pDNA is able to interact with microtubules in HeLa cells and traffick along them toward nucleus. Remarkably transfection efficiency of polyplexes is increased up to 77% in HeLa cells.
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Lucie Pigeon. Interactions moléculaires et cellulaires entre les protéines E3-14.7K, FIP-1 et les microtubules : application dans le transfert de gènes. Sciences agricoles. Université d'Orléans, 2012. Français. ⟨NNT : 2012ORLE2084⟩. ⟨tel-00912867⟩

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