AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS ...AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L lysine (PL), and LLO. Two marker genes, luciferase and β galactosidase in plasmids were complexed and administered in vitro to Huh7[ASG receptor (+) ] and SK Hep1[ASG receptor (-) ] cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated. RESULTS An LLO containing conjugate retained cell targeting specificity and membranolytic activity. In ASG receptor (+) cells, luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of β galactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies. CONCLUSIONS ASOR PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.展开更多
基金supported in part by agrant from the National Institutes of He alth,DK-42182(GYW)the Immune Response Cor por ation(GHW)the Herman Lopata Chair for He patitis Research.
文摘AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L lysine (PL), and LLO. Two marker genes, luciferase and β galactosidase in plasmids were complexed and administered in vitro to Huh7[ASG receptor (+) ] and SK Hep1[ASG receptor (-) ] cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated. RESULTS An LLO containing conjugate retained cell targeting specificity and membranolytic activity. In ASG receptor (+) cells, luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of β galactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies. CONCLUSIONS ASOR PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.
