Vectorisation synthétique d'acides nucléiques

La vectorisation synthétique d'acides nucléiques représente actuellement un enjeu déterminant pour le développement des traitements de pathologies héréditaires ou acquises. Les vecteurs synthétiques optimisés jusqu'à présent s'avèrent très efficaces in vitro pour la transfection de molécules d'ADN, cependant ils sont peu efficaces in vivo. Il est donc indispensable d envisager des stratégies de vectorisation innovantes améliorant l efficacité de transfection in vivo et adaptées aux différents acides nucléiques utilisés. Ainsi, une nouvelle classe de lipides cationiques dérivés de composés naturels, les aminoglycosides, a été développée pour la vectorisation d un nouveau type d acide nucléique, les siARN, qui sont des petits fragments d ARN double brin capables d inhiber l expression d un gène. Les dérivés lipidiques d aminoglycosides ont permis d obtenir une meilleure efficacité d inhibition de l expression d un gène, en utilisant des quantités de siARN plus faibles, par rapport à un lipide efficace pour la transfection des molécules d ADN. Dans une deuxième partie, une nouvelle classe de polymères amphiphiles, les copolymères à blocs, a été développée pour le transfert de gène dans les poumons. Les copolymères à blocs se sont montrés très efficaces pour transfecter un gène dans les cellules de l épithélium bronchique et alvéolaire des poumons de souris, et présentent aussi l avantage de pouvoir être administrer par aérosolisation. Enfin, nous avons synthétisé des matériaux hybrides originaux, basés sur l intercalation des molécules d ADN entre des feuillets inorganiques, les hydroxydes double lamellaires (HDL), labiles à pH acide. Le travail réalisé au cours de cette thèse a décrit la synthèse de nouvelles stratégies de vectorisation synthétique basée sur l utilisation de structures moléculaires originales.Synthetic vectorisation of nucleic acid has a promising future in the treatment of both hereditary and acquired lung diseases. Current synthetic vectors are efficient to deliver DNA molecule for in vitro studies, but their use for in vivo studies have been hampered by their low efficiency. Therefore, there is a need for developing alternative synthetic vectors to improve nucleic acid transfer in vivo and to investigate the vectorisation of different nucleic acid molecules. Thus, a new class of cationic lipid, derivatives from the family of the aminoglycosides natural compounds, have been developed for the transfection of a new type of nucleic acid, the siRNA. siRNA are small interfering RNA able to inhibit the expression of a gene. Lipidic aminoglycosides derivatives have shown their efficiency to deliver siRNA in cells, using smaller quantity than a lipid used for DNA molecules delivery. Then, a new class of amphiphilic block copolymers have been developed to improve gene transfer into the lung. Block copolymers had been very efficient to transfect epithelial cells in the bronchia and the alveolar areas. In a third part, we have synthesized a new supramolecular assemblies by formulation of DNA molecules with inorganic layered double hydroxides (LDH). Results have shown the in situ formation of LDH around DNA molecules characterized by a lamellar organization, where DNA molecules sandwiched between LDH. This work describe the synthesis of new vectorisation sytems based on the use of new molecular structure.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Biological pacemaker engineered by nonviral gene transfer in a mouse model of complete atrioventricular block.

International audienceWe hypothesized that a nonviral gene delivery of the hyperpolarization-activated HCN2 channel combined with the beta(2)-adrenergic receptor (ADRB2) would generate a functional pacemaker in a mouse model of complete atrioventricular block (CAVB) induced by radiofrequency ablation of the His bundle. Plasmids encoding HCN2 and ADRB2 mixed with tetronic 304, a poloxamine block copolymer, were injected in the left ventricular free wall (HCN2-ADRB2 mice). Sham mice received a noncoding plasmid. CAVB was induced 5 days later. Ventricular escape rhythms in HCN2-ADRB2 mice were significantly faster than in sham mice at day 15 after ablation and later. In HCN2-ADRB2 mice, QRS complexes were larger than in sham mice and characterized by abnormal axes. Immunostaining of GFP-HCN2 fusion protein showed an expression of HCN2 channel in left ventricular myocardium for at least 45 days after injection. In the mouse, CAVB induces progressive hypertrophy and heart failure leading to 50% mortality after 110 days. HCN2-ADRB2 mice survived 3 weeks longer than sham mice. Finally, beta-adrenergic input increased ventricular escape rhythms significantly more in HCN2-ADRB2 mice than in sham mice. In conclusion, nonviral gene transfer can produce a functional cardiac biological pacemaker regulated by sympathetic input, which improves life expectancy in a mouse model of CAVB

Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses