Structure et fonctionnement de vecteurs polymères pour la thérapie génique

Ce travail porte sur l étude physico-chimique de vecteurs polymères cationiques et neutres pour la thérapie génique. La structure et les interactions de mélanges ADN-pluronic L64, un polymère neutre, ont été étudiées par des techniques de diffusion de rayonnement. Contrairement aux vecteurs polymères classiques, le L64 n interagit pas de façon attractive avec l ADN et ne le compacte pas. Des interactions répulsives ont été mises en évidence entre les micelles de L64 et l ADN. Des mesures originales d électrophysiologie, montrent des interactions fortes entre les molécules de pluronic L64 et des membranes de lipides. Ces résultats indiquent que les molécules de L64 lors de la transfection permettraient aux molécules d ADN de pénétrer dans les cellules via un mécanisme de perforation. Concernant les polymères cationiques, la transition pelote-globule de longues chaînes individuelles d ADN induite par des polyéthylènimines de structure différentes par microscopie de fluorescence indique que cette transition dépend de la structure et peut influencer le polyplexe final utilisé en transfection.This work concerns the physicochemical study of cationic and neutral polymeric vectors for gene therapy. I studied the structure and the interactions of DNA-Pluronic L64 (a neutral polymer) mixtures by traditional biological and neutron scattering techniques. I showed that repulsive interactions are present between the DNA molecules and pluronic L64 micelles under physiological conditions. Original measurements of electrophysiology, enabled me to highlight strong interactions between the molecules of pluronic L64 and the lipid membranes. These results indicated that L64 molecules allow the DNA molecules, without interacting with it, to penetrate in the cells by a perforation mecanism. Measurements of fluorescence microscopy were set up to study the transition coil-globule of long individual DNA chains induced by linear or branched polyethylenimines, a reference cationic polymers in gene therapy. The results showed that this transition is a second second order phase transition and depends on the PEI structure.EVRY-BU (912282101) / SudocSudocFranceF

Stability of the I-motif Structure Is Related to the Interactions between Phosphodiester Backbones

The i-motif DNA tetrameric structure is formed of two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. The four phosphodiester backbones forming the structure define two narrow and wide grooves. The short interphosphate distances across the narrow groove induce a strong repulsion which should destabilize the tetramer. To investigate this point, molecular dynamics simulations were run on the [d(C2)]4 and [d(C4)]4 tetramers in 3′E and 5′E topologies, for which the interaction of the phosphodiester backbones through the narrow groove is different. The analysis of the simulations, using the Molecular Mechanics Generalized Born Solvation Area and Molecular Mechanics Poisson-Boltzmann Solvation Area approaches, shows that it is the van der Waals energy contribution which displays the largest relative difference between the two topologies. The comparison of the solvent-accessible area of each topology reveals that the sugar-sugar interactions account for the greater stability of the 3′E topology. This stresses the importance of the sugar-sugar contacts across the narrow groove which, enforcing the optimal backbone twisting, are essential to the base stacking and the i-motif stability. Tighter interactions between the sugars are observed in the case of N-type sugar puckers