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Transfert du CFTR par vecteurs de gènes dérivés des adénovirus ou par trogocytose de microparticules membranaires : mécanismes moléculaires et applications à la mucoviscidose

Abstract : The cystic fibrosis (CF) is a genetic disease due to mutations of the CFTR gene, resulting in the alteration of the Cl channel function carried by the transmembranal glycoprotein CFTR, and associated with severe pulmonary complications. Several recent studies led the medical and scientific community to reconsider the use of adenovirus serotype 5 (Ad5)-based vectors as CFTR gene transfer vectors in CF gene therapy. Not only immune response against the vector itself and the ansduced cells have been observed, but also Ad5-induced nondesired cytopathic effects. (1) In the first part of our study, we analyzed the cell entry and traficking of Ad5/F35, a chimeric vector consisting of serotype 5 capsid carrying serotype 35 fibers. We showed that the fibre protein is the major, if not only, determinant of the internalisation and entry pathway of Ad5/F35. Ad5/F35-GFP-CFTR, expressing the fusin protein GF-CFTR, was found (i) to be devoid of detectable cytopathic effect, (ii) efficiently transduced airway epithélial cells via the apical pole, and (iii) restore the Cl channel function in CF cells. Ad5/F35 therefore represents a CFTR gene transfer vector with a great potential for gene therapy of CF. (2) In the second part of our study, we have investigated an alternative strategy based on the transfer of the mature CFTR protein via trogocytosis. We hypothesized that microvesicles or microparticules (MP) issued from the cell membranes and released into the culture medium could transport and achieve the cell-to-cell transfer of CFTR channel cargo. We engineered donor cells for stable expression of GFP-tagged CFTR protein (GFP-CFTR), and showed that donor cell-issued MP were capable of delivering GFP-CFTR protein to recipient cell. However, the GFP-CFTR protein was only transferred by a limited population of MP (≤ 8 %), and was only transient (≤ 24h). In fact, the major population of MP transferred mRNAGFP-CFTR or polysomal thereof. Interestingly, the GFPCFTR protein newly synthesized from this mRNAGFP-CFTR was expressed at late times after transfer (≥ 48 h) but in a prolonged manner (≥ 10 jours). The Cl canal function after MP-mediated CFTR transfer is being evaluated. MP represent a novel type of CFTR vectors which can be produced by specifically designed autologous donor cells, and which would overcome most of the inconveniences of gene therapy using viral or nonviral vectors.
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https://tel.archives-ouvertes.fr/tel-00819319
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Submitted on : Tuesday, April 30, 2013 - 5:02:09 PM
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  • HAL Id : tel-00819319, version 1

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Gaëlle Gonzalez. Transfert du CFTR par vecteurs de gènes dérivés des adénovirus ou par trogocytose de microparticules membranaires : mécanismes moléculaires et applications à la mucoviscidose. Médecine humaine et pathologie. Université Claude Bernard - Lyon I, 2011. Français. ⟨NNT : 2011LYO10261⟩. ⟨tel-00819319⟩

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