Taniyama Yoshiaki, Tachibana Katsuro, Hiraoka Kazuya, Namba Tsunetatsu, Yamasaki Keita, Hashiya Naotaka, Aoki Motokuni, Ogihara Toshio, Yasufumi Kaneda, Morishita Ryuichi
Department of Geriatric Medicine, Osaka University, Graduate School of Medicine, Osaka, Japan.
Circulation. 2002 Mar 12;105(10):1233-9. doi: 10.1161/hc1002.105228.
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.
尽管病毒载体系统能有效地将外源基因转染至血管，但在人类基因治疗方面仍存在安全问题。在本研究中，我们通过使用高频、低强度超声照射来研究一种新型非病毒载体系统转染血管的可行性。
将荧光素酶质粒与回声对比微泡（Optison）混合或不混合，利用超声将其转染至培养的人血管平滑肌细胞（VSMC）和内皮细胞（EC）中。有趣的是，在两种用Optison处理的细胞类型中，荧光素酶活性均显著增加。然后，我们通过治疗性超声将与Optison混合的荧光素酶质粒转染至大鼠动脉。转染后两天，用荧光素酶基因与Optison及超声处理的颈动脉中的荧光素酶活性显著高于单独使用质粒处理的情况。此外，我们将一种抗癌基因（p53）质粒转染至球囊损伤后的颈动脉，作为再狭窄基因治疗的模型。转染后两周，通过超声用野生型p53质粒与Optison复合处理的大鼠，其内膜与中膜面积比与对照组相比显著降低，同时p53蛋白显著增加。在用超声和Optison转染质粒DNA的血管中未检测到明显的毒性，如炎症。
总体而言，我们证明了使用Optison的超声转染方法可提高裸质粒DNA转染至血管的效率，且无明显毒性。使用该方法转染p53质粒对于无病毒载体系统的安全临床基因治疗应是有用的。
