Lyophilisation et concentration de complexes chitosane/ADN et chitosane/ARNic pour la thérapie génique

RESUME Beaucoup de ressources ont ete investies au cours des dernieres decennies pour developper des vecteurs de livraison de genes polymeriques securitaires et efficaces pour des applications cliniques. Le chitosane (CS), un polysaccharide cationique naturel, permet de condenser l’ADN ou l’ARN interferent court (ARNic) pour former des polyplexes dont l’efficacite de transfection elevee a ete demontree in vitro et in vivo. Afin d’etre utilisee en clinique, ces formulations doivent toutefois atteindre des doses therapeutiques; elles doivent etre adaptees pour un usage intraveineux; et elles doivent etre stabilisees pour le transport et l’entreposage. Les objectifs de la these etaient: d’identifier des formulations biocompatibles de complexes CS/ADN et CS/ARNic pouvant etre lyophilisees et concentrees, tout en preservant leurs proprietes et en demeurant isotoniques; de developper des compositions lyophilisees de polyplexes hemocompatibles ayant un pH neutre; et d’optimiser les cycles de lyophilisation des formulations. Une premiere etude, menee sur des polyplexes CS/ADN prepares a un ratio amine/phosphate (N/P) de 5, a permis de determiner que l’ajout de 0.5% m/V de sucrose, dextran 5 kDa ou trehalose, combine a 3.5 mM histidine (pH 6.5), etait necessaire pour preserver le pH des formulations et eviter l’agregation des complexes pendant la lyophilisation. Ces compositions pouvaient ensuite etre rehydratees a une concentration 20 fois plus elevee (~1 mg d’ADN/mL), tout en ayant un pH et une tonicite quasi physiologiques. Les formulations concentrees ont genere des polyplexes ayant des diametres inferieurs a 200 nm, des indices de polydispersite (PDI) inferieurs a 0.32, et des potentiels zeta superieurs a 19 mV. Leurs taux de transfection et d’expression de la luciferase etaient superieurs ou egaux a 65% et 75% des polyplexes fraichement prepares sans excipients respectivement. Finalement, les compositions n’etaient pas cytotoxiques pour la lignee cellulaire renale HEK293 aux concentrations evaluees. Ces connaissances ont ensuite ete adaptees pour la formulation des polyplexes CS/ARNic. L’analyse de l’influence des tampons sur les compositions a demontre que les proprietes physicochimiques des polyplexes varient selon la valence de leurs contre-ions dans le tampon, selon la force ionique, et selon le pH des formulations. Les polyplexes prepares a N/P = 2 en presence d’acide maleique ou de phosphate de sodium agregeaient lorsque le pH et/ou la force ionique augmentaient; les resultats optimaux ont ete obtenus avec histidine, soit en absence de contre-ions anioniques divalents. La concentration des tampons compatibles a ete optimisee pour----------ABSTRACT Research to develop safe and efficient non-viral gene delivery vectors for clinical applications has gained momentum in recent years. Chitosan (CS) is a natural cationic polysaccharide which has been successfully tailored to self-assemble with DNA or small interfering RNA (siRNA) to form polyelectrolyte complexes with high in vitro and in vivo transfection efficiencies. Despite their great therapeutic potential, CS/DNA and CS/siRNA polyplex formulations require further improvement for successful clinical applications: their concentration should be increased to reach therapeutic dosages, they should be optimized for intravenous administration, and they should be stabilized for shipping and long term storage. The goals of the thesis were: to identify biocompatible CS/DNA and CS/siRNA formulations that can be lyophilized and rehydrated to higher concentrations while preserving their properties and being isotonic; to develop hemocompatible, neutral pH, lyophilized polyplex compositions; and to optimize freeze-drying cycles for these compositions. An initial study done with CS/DNA polyplexes, prepared at amine to phosphate (N/P) ratio of 5, showed that 3.5 mM histidine buffer (pH 6.5), combined with one of 0.5 % wt/vol sucrose, 5kDa dextran, or trehalose, was required in the formulations to prevent complex aggregation during freeze-drying. These compositions could then be rehydrated to final concentrations up to 20-fold higher (~ 1 mg of DNA/mL), while maintaining near physiological pH and tonicity. The resulting polyplexes were predominantly spherical, with diameters below 200 nm, polydispersity indexes (PDI) below 0.32, and zeta potentials above +19 mV. Rehydrated formulations had transfection efficiencies and luciferase expression levels no less than 65% and 75% of fresh polyplexes without excipients respectively. They did not affect the viability of the HEK 293 kidney cell line. This knowledge was then adapted to CS/siRNA systems. Analysis of the influence of buffers on polyplexes showed three buffer parameters affect their physicochemical properties, namely the valency of their counterions, their ionic strength, and the formulation pH. Polyplexes prepared at N/P = 2 in maleic acid or sodium phosphate aggregated when pH and/or ionic strength increased, whereas optimal results were obtained with histidine, in absence of divalent anionic counterions. Compatible buffers were then optimized for freeze-drying of uncoated and hyaluronic acid (HA)-coated polyplexes in 0.5% wt/vol trehalose. Compositions prepared at pH 6.5 could be lyophilised with 1 mM histidine, whereas HA-coated polyplexes prepared at pH 7 had to be