Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

227 págs.

[EN] Over the past few years and due to biotechnology development, gene therapy has emerged as a promising therapeutic tool because of the use of exogenous nucleic acids as active ingredients. These nucleic acids, i.e. Deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), can be delivered into the organism in order to modulate the expression of proteins modified in particular diseases.These acquired or genetic diseases include pathologies such as cancer, AIDS, neurological disorders or autoimmune diseases. Ocular gene therapy is one of the most investigated fields and its progress lies, among other things, in the easy accessibility into the eye and the convenient methods for its evaluation. Thus, several research groups use gene therapy as alternative to treat diseases such as retinitis pigmentosa, macular degeneration and X-linked juvenile retinoschisis. The vast majority of these studies are carried out using viral vectors to deliver genetic material, as they have proved to be the most effective to date. Nevertheless, the safety-related risks that involve their use have triggered the development of non-viral vectors, and the improvement of their efficacy has become a key challenge in gene therapy. In order to design non-viral vectors, an understanding of the extra and intracellular barriers conditioning the transfection process is needed: interaction and subsequent passage through cellular membrane, intracellular trafficking, resistance against degradation, genetic material release, passage through nuclear membrane and finally, expression of the protein codified by the plasmid that vector transports.In the present work hybrid gene delivery systems have been developed and evaluated. These systems, based on solid lipid nanoparticles (SLN), also include compounds of different nature to favor the transfection process by mans of several mechanisms. Formulations were characterized in terms of size, surface charge, protection capacity against DNAses and plasmid release from the nanoparticle. Furthermore, “in vitro” transfection capacity, cellular uptake, endocytosis mechanisms and intracellular trafficking were studied by means of confocal laser microscopy and flow cytometry. These processes were carried out over two cell lines (ARPE-19 and HEK-293) and were related to transfection capacity of the formulations developed.Firstly, the influence of a peptide, protamine, in the transfection capacity of SLN was evaluated. The presence of protamine in SLN increased the “in vitro” transfection capacity in ARPE-19 cells, with respect to SLN without protamine, but it caused a decrease in HEK-293 cells. It was observed that this different behavior is conditioned by the cellular uptake mechanism of nanoparticles, which depends on cell line as well as vector composition. Next, a polysaccharide, dextran, was included in the formulation. When lipid nanoparticles were prepared with protamine and the dextran, a high transfection capacity was achieved in ARPE-19 cells with the plasmid that codifies green fluorescent protein (EGFP) as well as the one that codifies retinoschisin, a protein related to X-linked juvenile retinoschisis. Once the vector was optimized by means of “in vitro” studies, the “in vivo” evaluation was conducted. For that purpose, a vector prepared with SLN, protamine, dextran and the plasmid that codified EGFP was administrated by different ocular routes in rats. After intravitreal injection, retina ganglion cells were mainly transfected, and when administrated by subretinal injection, retinal pigment epithelium cells as well as photoreceptors were the most transfected cells. After topical application, the vector was also able to transfect corneal cells. Finally, transfection capacity after intravenous administration into mice was also evaluated, detecting green fluorescent protein in liver, spleen and lung; the expression was maintained for at least seven days.