Taniyama Y, Tachibana K, Hiraoka K, Aoki M, Yamamoto S, Matsumoto K, Nakamura T, Ogihara T, Kaneda Y, Morishita R
Department of Geriatric Medicine, Graduate School of Medicine, Osaka University, Suita, Japan.
Gene Ther. 2002 Mar;9(6):372-80. doi: 10.1038/sj.gt.3301678.
Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.
尽管使用裸质粒 DNA 或腺病毒载体转染血管生成生长因子来刺激血管生成的临床试验已取得成功，但人类基因治疗仍存在一些未解决的问题，如转染效率低和安全性问题。从这个角度来看，有必要开发安全有效的新型非病毒基因传递方法。由于治疗性超声可诱导细胞膜通透性增加，超声照射可能会提高裸质粒 DNA 进入骨骼肌的转染效率。因此，我们在体外和体内实验中研究了使用超声与超声造影微泡（Optison）照射对裸质粒 DNA 转染效率的影响。首先，我们研究了超声介导的裸质粒 DNA 转染骨骼肌细胞的可行性。将与 Optison 混合或未混合的荧光素酶质粒用超声（1MHz；0.4W(2)）处理 30 秒后转染至培养的人骨骼肌细胞中。有趣的是，用 Optison 处理的细胞中荧光素酶活性显著增加，而未用 Optison 处理的细胞几乎检测不到荧光素酶活性（P<0.01）。电子显微镜显示细胞表面短暂形成小孔（小于 5 微米），这可能解释了转基因快速进入细胞的现象。接下来，我们研究了使用超声与 Optison 将裸质粒 DNA 体内转染至骨骼肌的效率。转染两天后，与单独使用质粒相比，使用超声与 Optison 转染的骨骼肌中的荧光素酶活性显著增加了约 10 倍。β-半乳糖苷酶染色也证实了成功转染。最后，我们在兔缺血模型中使用超声 - Optison 方法研究了使用裸肝细胞生长因子（HGF）质粒进行治疗性血管生成的可行性。转染五周后，与单独使用 HGF 质粒相比，使用超声与 Optison 转染的兔的血管造影评分和毛细血管密度显著增加（P<0.01），同时血流量和血压比也显著增加（P<0.01）。总体而言，超声与 Optison 转染方法提高了裸质粒 DNA 在体内和体外的转染效率。超声 - Optison 方法转染 HGF 质粒可能有助于在无病毒载体系统的情况下安全地进行临床基因治疗以治疗外周动脉疾病。
