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 beta 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 target-ing 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 betagalactosidase 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.
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 beta 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 target-ing 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 betagalactosidase 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.
Authors: M Feng; W H Jackson; C K Goldman; C Rancourt; M Wang; S K Dusing; G Siegal; D T Curiel Journal: Nat Biotechnol Date: 1997-09 Impact factor: 54.908