Qiao Chunping, Koo Taeyoung, Li Juan, Xiao Xiao, Dickson J George
Division of Molecular Pharmaceutics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Methods Mol Biol. 2011;807:119-40. doi: 10.1007/978-1-61779-370-7_5.
Adeno-associated virus (AAV) is the most promising gene delivery vehicle for muscle-directed gene therapy. AAV's natural tropism to muscle cells, long-term persistent transgene expression, multiple serotypes, as well as its minimal immune response have made AAV vectors well suited for muscle-directed gene therapy. AAV vector-mediated gene delivery to augment muscle structural proteins, such as dystrophin and sarcoglycans, offers great hope for muscular dystrophy patients. In addition, muscle can be used as a therapeutic platform for AAV vectors to express nonmuscle secretory/regulatory pathway proteins for diabetes, atherosclerosis, hemophilia, cancer, etc. AAV vector can be delivered into both skeletal muscle and cardiac muscle by means of local, regional, and systemic administrations. Successful animal studies have led to several noteworthy clinical trials involving muscle-directed gene therapy. In this chapter, we describe the basic methodology that is currently utilized in the area of AAV-mediated muscle-directed gene therapy. These methods include vector delivery route, vector dosage, detection of transgene expression by immunostaining and western blot, determination of vector copy numbers and quantification of mRNA expression, as well as potential immune responses involved in AAV delivery. Technical details and tips leading to successful experimentation are also discussed.
腺相关病毒（AAV）是用于肌肉定向基因治疗最有前景的基因递送载体。AAV对肌肉细胞的天然嗜性、转基因的长期持续表达、多种血清型以及其最小的免疫反应，使得AAV载体非常适合肌肉定向基因治疗。AAV载体介导的基因递送以增强肌肉结构蛋白，如抗肌萎缩蛋白和肌聚糖，为肌营养不良患者带来了巨大希望。此外，肌肉可作为AAV载体的治疗平台，用于表达糖尿病、动脉粥样硬化、血友病、癌症等疾病的非肌肉分泌/调节途径蛋白。AAV载体可通过局部、区域和全身给药方式递送至骨骼肌和心肌。成功的动物研究已促成了多项涉及肌肉定向基因治疗的重要临床试验。在本章中，我们描述了目前在AAV介导的肌肉定向基因治疗领域中使用的基本方法。这些方法包括载体递送途径、载体剂量、通过免疫染色和蛋白质印迹检测转基因表达、确定载体拷贝数和定量mRNA表达，以及AAV递送中涉及的潜在免疫反应。还讨论了导致成功实验的技术细节和技巧。
