CHARACTERIZATION OF LIPOSOME-MEDIATED GENE DELIVERY - EXPRESSION, STABILITY AND PHARMACOKINETICS OF PLASMID DNA

We have characterized a new synthetic gene delivery system, termed DLS , which may be suitable for systemic gene therapy. DLS constitutes a l ipopolyamine and a neutral lipid and associated plasmic DNA in the for mation of lamellar vesicles (DSL-DNA). The ratio of and lipid to DNA a s well as the method of preparation were optimized to a high in vitro transfection efficiency compared with reported for cationic lipid syst ems. DLS-DNA showed a rapid cellular uptake and distribution in the cy toplasmic and nuclear (especially the nucleoli) compartment as determi ned by laser-assisted confocal microscopy. There was little or no plas mid DNA degradation over a period of 20 min, relatively slow plasma cl earance, and effective and rapid cellular uptake of DLS-DNA following intravenous administration in mice. Supercoiled plasmid DNA could be d efected in blood cells up to 1 h after injection. Systemic administrat ion of DLS-DNA yielded transgene expression in mouse tissues, such as in lung or liver. The ratio of DLS:DNA and the procedure used to form DLS-DNA affected both the level cellular specificity of expression of a luciferase reporter gene showing that in vitro transfection efficien cy of DLS-DNA formulations cannot be easily extrapolated to an in vivo setting. Optimization of the formulation of a PNA delivery system was critical to obtain a defined structure resulting in a preparation wit h high reproducibility and stability, greater homogeneity of particle size and high efficacy following systemic gene transfer. In addition, the DLS system may be formulated for specific target tissues and may h ave a wide range of applications for gene therapy.