677. Polycationic Liposome-Mediated Extracellular Superoxide Dismutase Gene Delivery Leads to High Levels of the Transgene Expression and Prevents Acute Liver Injury in Mice

Abstract

BACKGROUND: We have developed a formulation of polycationic liposomes that has been shown to be non-toxic, highly stable in the bloodsteam, and very effective in liver gene transfer in mice (Gene Therapy 2003; 10:180–187). Extracellular superoxide dismutase (EC-SOD) inhibits the generation of superoxide anions in the interstitial space of tissues, and may play a role in minimizing oxidative stress in liver injury. The aim of the present study is to investigate whether our polycationic liposome-mediated human EC-SOD gene delivery will protect against lipopolysaccharide (LPS)-induced liver toxicity in D-galactosamine (GalN)-sensitized mice. METHODS: Polycationic liposomes were generated from polycationic lipid (PCL) and cholesterol (Chol). Lipoplexes were formed by complexing liposomes with control plasmid (pEGFP-C1) or EC-SOD plasmid (pEGFP-C1-ECSOD) before use. Mice were injected with thyroid hormone (T3, 4 mg/kg, s.c.) two days before lipoplexes were injected via the portal vein. One day following lipoplex injection, mice were treated with GalN (500 mg/kg, i.p.) plus LPS (25 mg/kg, i.p.). Serum alanine aminotransferase (ALT), SOD activity, liver histology, glutathione (GSH) content and lipid peroxidation were evaluated one day after GalN/LPS exposure. Human EC-SOD gene expression in mouse liver tissue was determined by quantitative RT-PCR two days after lipoplex injection. RESULTS: somes and the control lipoplexes were stable in their size for three months at 4°C. Injection of T3, PCL-Chol liposomes or control lipoplexes did not cause a significant change in serum ALT levels. Real time quantitative RT-PCR analysis showed that human EC-SOD mRNA levels in mouse liver tissue in EC-SOD lipoplex-injected group were 55-fold higher than saline, liposome or control lipoplex controls when mouse -actin was employed as a house-keeping control gene. Serum ALT levels in animals receiving portal vein injections of EC-SOD lipoplexes plus GalN/LPS exposure were much lower than in those receiving normal saline, liposomes alone, or control lipoplexes plus GalN/LPS exposure (62583 vs. 150865, 153194, 1880520 units/ml, p<0.01). Liver histology showed much less hepatocyte death in EC-SOD lipoplex-treated group than liposome- or control lipoplex-treated groups. Serum SOD activity in animals receiving injection of EC-SOD lipoplexes were higher than in those treated with liposomes only, or control lipoplexes (28.8 3.3 vs. 15.2 0.9, 17.4 3.4 units/ml, p<0.05). Liver GSH content in animals receiving EC-SOD lipoplex injection was preserved with less lipid peroxidation, as indicated by a lower level of malondialdehyde content. CONCLUSIONS: liposome-mediated EC-SOD gene delivery leads to the marked transgene expression, and attenuates acute liver injury due to reduced oxidative stress in GalN/LPS-exposed mice. To our knowledge, this is a first report which demonstrates that polycationic liposome-mediated EC-SOD gene delivery to the liver represents a potential therapy for hepatotoxin-induced liver injury.