Lewkowicz, Nina Maria; (2001) High-yield incorporation of plasmid DNA into liposomes: characterisation and transfection efficiency studies. Doctoral thesis (Ph.D.), University College London.

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Abstract

The aim of the thesis was to investigate the potential use of liposomes as a carrier for DNA delivery in vitro and in vivo. In the first stage, we examined the incorporation of plasmid DNA pGL2 encoding luciferase and pRc/CMV HBS plasmid DNA encoding HBsAg (S region; ayw subtype) into liposomes prepared by the mild method of dehydration-rehydration (DRV liposomes). The prepared systems were characterised in terms of vesicle size, lipid composition and surface charge [e.g. vesicles composed of PC and DOPE alone or supplemented with anionic (PS or PG) or cationic lipids (DOTMA, SA, DC-Chol or DOTAP)], and stability. The ability of DRV liposomes and microfluidised DRV liposomes to protect their nucleic acid content from deoxyribonuclease attack was also analysed and compared with naked DNA or DNA complexed with preformed cationic liposomes. Furthermore, the transfection activity was assessed by using pGL2 as model DNA incorporated into various types of liposomes. Next, the effect of various DRV liposomal formulations incorporating plasmid DNA pRc/CMV HBS on both humoral and cellular immunity responses to the encoded antigen was determined and compared with responses from naked DNA or DNA complexed with preformed DRV. DNA immunisation dose response studies were carried out intramuscularly using Balb/c mice. In the final part of the thesis, we assessed the effect of various routes of injection on eliciting immune responses to the antigen encoded by pRc/CMV-HBS, using both inbred (Balb/c) and outbred (T/O) mice. Liposomal plasmid DNA was found to retain its structural integrity and to transfect COS-7 cells in vitro in relation to the size and surface charge of the vesicles. Moreover, our studies suggest that plasmid DNA incorporated into cationic DRV liposomes under the conditions described is more effective than plasmid DNA in saline formulation in inducing both humoral and cellular immunity.

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