Gerber, SandrineNicolle, Laura Camille Louise2022-07-182022-07-182022-07-18202210.5075/epfl-thesis-9821https://infoscience.epfl.ch/handle/20.500.14299/189435Gene therapy offers the possibility to treat or even cure diseases originating from genetic defects by introducing a therapeutic gene in target cells or correcting the initial defective gene. Amongst different options, non-viral vectors rely on the delivery of such genes using vehicles composed of lipids, inorganic nanoparticles or polymers. These systems are designed to protect the genetic material, while efficiently delivering it to the cells of interest. The initial backbone can be complemented with various components to help it overcome the many physiological barriers it may encounter during its journey. Amongst polymers, chitosan is a polysaccharide presenting many advantages for such purpose. Besides its biocompatibility and biodegradability, its potential for functionalization, thanks to numerous primary amines and hydroxyl groups, make it a backbone of choice for the preparation of a polymeric delivery vehicle. This work describes the covalent functionalization of chitosan with other polymers, peptides, and small molecules to later form polymeric nanoparticles condensing therapeutic DNA. Each component brings additional features to the initial system, thus finely tuning it for biological applications. The final system aims at treating liver inherited diseases such as ornithine transcarbamylase deficiency and phenylketonuria. This manuscript first exposes the preparation of a chitosan-based cationic system able to properly condense anionic DNA through electrostatic interactions. Due to its excellent DNA condensation properties, polyethylenimine was grafted to chitosan via a succinyl linker. The first in vitro investigations confirmed the potential of the resulting core system, but in vivo experiments highlighted toxicity coming from the remaining cationic charge on the nanoparticles. In order to better shield this cationic charge, bifunctional polyethylene glycol chains terminated by carboxylic acid moieties were grafted to chitosan amines. The resulting polymer was used to coat the first polymeric system through electrostatic interactions between the carboxylate groups and the remaining free amines of the chitosan-polyethylenimine derivative. The newly formed core-shell complex showed sustained cell viability in vitro and induced higher transfection efficiency of the gene as compared to the core system alone. However, its in vivo evaluation via retrograde intrabiliary infusion did not confirm such results: although transfection efficiency was retained, the core-shell complex did not significantly decrease the toxicity previously reported. The last part of this work includes the decoration of the core-shell system with targeting ligands making possible to perform in vivo assays via intravenous injection, for which the developed polymeric systems did not induce toxicity in preliminary experiments. So far, only a proof-of-concept with folic acid, an anti-cancer targeting ligand, was achieved. To do so, the shell polymer was further functionalized with folic acid-derived polyethylene glycol chains through strain-promoted azide-alkyne cycloaddition. The same strategy shall be applied in the near future with a liver-specific targeting ligand. Lastly, the improvement of the system's cell-penetration features was tackled by the grafting asparagusic acid-derived polyethylene glycol chains following the same strategy than for folic acid.enchitosanliver-inherited diseasenon-viral gene deliverypolyethyleniminepolymeric vehiclepolyethylene glycolcell-penetrating peptidesfolic acidasparagusic acidthesis::doctoral thesis