PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.
PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.
Authors: P Erbacher; T Bettinger; P Belguise-Valladier; S Zou; J L Coll; J P Behr; J S Remy Journal: J Gene Med Date: 1999 May-Jun Impact factor: 4.565
Authors: Michiel Voskuil; Niels van Royen; Imo E Hoefer; Randolph Seidler; Brian D Guth; Christoph Bode; Wolfgang Schaper; Jan J Piek; Ivo R Buschmann Journal: Am J Physiol Heart Circ Physiol Date: 2002-12-27 Impact factor: 4.733