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      Local delivery of plasmid DNA into rat carotid artery using ultrasound.


      Ultrasonics, Albumins, administration & dosage, Animals, Carotid Arteries, drug effects, metabolism, pathology, Carotid Stenosis, prevention & control, Catheterization, Cells, Cultured, DNA, Complementary, Endothelium, Vascular, ultrastructure, Feasibility Studies, Fluorocarbons, Gene Expression, Gene Transfer Techniques, Genes, Reporter, Humans, Luciferases, biosynthesis, genetics, Male, Muscle, Smooth, Vascular, Plasmids, Rats, Rats, Sprague-Dawley, Transfection, methods, Tumor Suppressor Protein p53

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          Although viral vector systems are efficient to transfect foreign genes into blood vessels, safety issues remain in relation to human gene therapy. In this study, we examined the feasibility of a novel nonviral vector system by using high-frequency, low-intensity ultrasound irradiation for transfection into blood vessels. Luciferase plasmid mixed with or without echo contrast microbubble (Optison) was transfected into cultured human vascular smooth muscle cells (VSMC) and endothelial cells (EC) with the use of ultrasound. Interestingly, luciferase activity was markedly increased in both cell types treated with Optison. We then transfected luciferase plasmid mixed with Optison by means of therapeutic ultrasound into rat artery. Two days after transfection, luciferase activity was significantly higher in carotid artery transfected with luciferase gene with Optison and ultrasound than with plasmid alone. In addition, we transfected an anti-oncogene (p53) plasmid into carotid artery after balloon injury as a model of gene therapy for restenosis. Two weeks after transfection, the intimal-to-medial area ratio in rats transfected with wild-type p53 plasmid complexed with Optison by means of ultrasound was significantly decreased as compared with control, accompanied by a significant increase in p53 protein. No apparent toxicity such as inflammation could be detected in blood vessels transfected with plasmid DNA with ultrasound and Optison. Overall, we demonstrated that an ultrasound transfection method with Optison enhanced transfection efficiency of naked plasmid DNA into blood vessels without any apparent toxicity. Transfection of p53 plasmid with the use of this method should be useful for safe clinical gene therapy without a viral vector system.

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