Investigation of intracellular plasmid DNA delivery by encapsulated DNA particles and DNA-lipid complexes

Transfection of eukaryotic cells utilizing cationic lipid complexed to plasmid DNA has become an increasingly important model for study and development of intracellular gene delivery. The mechanisms by which cationic lipids mediate intracellular delivery of plasmid DNA are currently not well understood. It has been proposed that DNA-lipid complexes transfect cells through a series of events which includes interaction of the DNA-lipid complex with the cell surface (Friend et al. 1996), entry of the DNA-lipid complex into the cell by way of endocytosis (Zabner et al. 1995, Friend et al. 1996, Zhou et al. 1994), followed by dissociation of cationic lipid from the plasmid DNA (Xu and Szoka, 1996) and its subsequent transport from the cytoplasm to the nucleus where gene expression occurs. However limitations of gene delivery systems like DNA-lipid complexes have led to the development of potentially more versatile encapsulated DNA particles that exploit the ability of biological membranes to act as protective barriers to exterior nucleases and DNases found in the serum. Hence the lipid can act as a shield, protecting the plasmid DNA until it has the opportunity to reach its target site in vitro or in vivo. The aim of this thesis is to understand how structural differences between the DNA-lipid complexes and encapsulated DNA-cationic lipid particles can affect the rate of plasmid DNA uptake into the cell, the level of gene expression and the persistence of AAV vectors in target cells over time. Initially encapsulated plasmid DNA samples were characterized utilizing various labeled lipids and labeled plasmid DNA to determine a purification profile by sucrose density gradients. Purification of the encapsulated DNA sample produced two distinct populations, empty lipid particles and encapsulated DNA particles. The ratio of carrier lipid to plasmid DNA and the ratio of cationic lipid to plasmid DNA were unique for both the empty liposomes and the DNA-lipid particles. Furthermore the ratio of carrier lipid to cationic lipid was also found to be unique for the two populations observed. Picogreen fluorescence analysis was used to corroborate the presence of the encapsulated DNA peak. From these results, it was concluded that two unique populations, empty liposomes and encapsulated DNA particles were present in the original encapsulated DNA sample. Plasmid DNA uptake into 293 cells and transfection levels for purified, entrapped DNAcationic lipid particles and to DNA-lipid complexes were examined. The plasmids pCMV- P , pAAV- P and pAAV- P + Rep were transfected into cells either by complexing the plasmid DNA to pre-formed liposomes or by encapsulating the plasmids. The levels of P - galactoside activity were considerably lower when utilizing the encapsulated DNA particles compared to the DNAlipid complexes. Accordingly the level of cellular uptake of the plasmids pCMV-P and pWP-19 delivered by encapsulated DNA particles was also significantly lower compared to the DNAlipid complexes. The levels of gene expression obtained with the integrating vectors pAAV- P + Rep produced slightly higher levels of gene expression in comparison to the non-integrating vectors pCMV- p , while the plasmid pAAV- p produced lower or similar levels of gene expression in comparison to pCMV- p . Cellular uptake levels of the integrating vector pWP-19 was significantly higher than the cellular uptake level of pCMV- p . This difference was attributed to the retention of the plasmid over time. Finally the number of G418 resistant colonies obtained by the two transfection methods was examined. The number of colonies obtained using encapsulated DNA particles was approximately 10 fold lower than the DNA-lipid complexes. The number of colonies obtained with the newly constructed vector pWP-19.5, which lacks 1 ITR site resulted in the production of 30 % fewer colonies in comparison to the 2 ITR vector construct.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat