The role of the carrier and the endocytic pathway in non-viral gene delivery

Gene therapy offers promise for the treatment of both inherited and acquired diseases through the introduction of genetic material into target cells. The primary challenge for gene therapy is to develop a safe and efficient method for the delivery of therapeutic genetic material to the specific intracellular target. Non-viral carriers have received significant attention because of their potential to overcome the limitations of viral-based systems. However, their relatively low efficacy is a major obstacle to their clinical application. A thorough understanding of the key factors affecting the gene delivery process will provide clues on how to develop more effective carriers. This thesis focuses on the role of the carrier and the endocytic pathway in non-viral gene delivery, and also suggests improvements in the experimental methodology that would make it possible to obtain more reliable results in nanoparticle uptake studies. More effective carriers for gene delivery are very much needed. We tested the novel pentaspheric lysine-based dendrimer and its analog, modified with fatty acid residues, for their gene delivery capacity. We demonstrated that despite their relatively low in vitro transfection efficacy, lysine dendrimers have good plasmid DNA (pDNA) binding and protective properties, and can therefore be used as the basis for the development of more effective carriers. A detailed understanding of the cellular kinetics of gene delivery systems is critical to the further development of more effective carriers. We studied the impact of the carrier and of the endocytic pathway on cell uptake and the intracellular processing of genetic material (pDNA). The highly sensitive method of quantitative real-time PCR was applied to the study of the intracellular kinetics of pDNA introduced by the cationic polymer PEI, cationic lipid DOTAP, and CaP precipitates at multiple time points after transfection. The results obtained indicate that the carrier affects the cell uptake and the intracellular kinetics, and therefore predetermines the main transfection-limiting step. Furthermore, we demonstrated the important role of the post-nuclear processes in efficient non-viral gene delivery. The impact of a specific endocytic pathway was studied by the inhibition of either the clathrinor dynamin-mediated endocytosis. Selective blockage of endocytosis was achieved by two approaches, namely the genetically manipulated cell lines and the chemical inhibitors of endocytosis. Analysis of the intracellular kinetics of pDNA in the genetically blocked cells revealed that neither the amount of pDNA taken up by the cell, nor the intracellular pDNA elimination, but the amount of pDNA delivered to the nucleus was indicative of the significance of the particular pathway in the resulting efficacy of the carrier. A comparison of chemical and genetic means for blocking endocytosis revealed the limitations of both these methods. A careful optimization of the method and the use of several alternative approaches is recommended in order to obtain more reliable data. We suggest that the characterization of in vitro cell models for the expression and activity of specific endocytic pathways (endocytic profiling) would facilitate the interpretation of the data obtained in nanoparticle uptake studies. Endothelial and epithelial cells are widely utilized in such studies because they form substantial barriers en route from the administration site to the target tissue. We performed endocytic profiling of