Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

Hepatitis C virus (HCV) causes a significant health problem worldwide due to the lack of effective vaccines. It has been recognized that a rapid, vigorous, and broadly targeted cell-mediated immune response (Th1-like) is often associated with the clearance of HCV infections. DNA vaccines represent a promising means for HCV vaccination because they tend to induce a Th1-biased cell-mediated response in the host cell. Currently, the delivery of DNA vaccine for HCV in large animals as well as in humans is not as effective as in small animals. Nano delivery systems would be a promising approach to overcome this problem. Carbon nanotubes (CNTs) have been extensively studied for delivering drugs, proteins, peptides, and nucleic acids including plasmid DNA to cells and organs with varying degrees of success, but few of them have been applied to DNA vaccine for HCV.<p> This thesis presents a study of using functionalized CNTs (f-CNTs) to improve the efficacy of plasmid DNA vaccine delivery for HCV. First, CNTs were functionalized via 1,3-dipolar cycloaddition reaction with the appropriate amino acids and aldehydes. NMR and TEM results suggested that the CNTs were successfully functionalized and became soluble in water. Then plasmid DNAs which encode green fluorescence protein reporter gene, luciferase reporter gene, and HCV core protein, respectively, were delivered into human hepatoma cells via calcium phosphate precipitation method, f-CNT delivery system, and a combination of f-CNT and calcium phosphate method, respectively. The result showed that f-CNTs, in combination with the calcium phosphate method, significantly enhanced the gene expression in human hepatoma cells.<p> Consequently, this study concludes that the f-CNT can significantly enhance gene expression in liver cells conferred by a plasmid DNA when combined with calcium phosphate precipitation method. Even though the mechanisms of this enhancement await further investigation, the results of this thesis may have important implications in developing DNA vaccines for infectious diseases in general and for hepatitis C in particular.

Transfection efficiencies of several polymeric gene carriers were compared and correlated quantitatively to the amounts of cellular accumulation of plasmid DNA and to the expression of mRNA by quantitative real time PCR. Three cationic methacrylate polymer systems with similar chemical structure were used in this study, namely: poly(dimethylamino)ethyl methacrylate (PDMA) homopolymer, PEO-b-PDMA copolymer and PEO-b-poly(diethylamino)ethyl methacrylate (PEO-b-PDEA) copolymer. Despite their similar chemical structures, their transfection efficiencies were significantly different. PEO-b-PDEA copolymer was significantly less efficient as gene carrier compared to both PDMA and PEO-b-PDMA systems. Results from quantitative real-time polymerase chain reaction (real-time PCR), cytotoxicity and Zeta potential measurements showed correlations between the physical properties of the polymers and the efficiencies of cellular uptake of the transgene and transfections. In the case of PEO-b-PDEA system, cytotoxicity was due primarily to the excess polymers that did not participate in the DNA binding. In addition, the inability of the polymer/DNA complexes to interact with cell effectively was identified as the main barrier for high efficiency of transfection. This study demonstrated that the use of quantitative real-time PCR in combination with other physical characterization techniques can provide greater insights into the transfection barrier at different cellular levels. / Singapore-MIT Alliance (SMA)