Gene Therapy in Skeletal Muscle Mediated by Adeno-Associated Virus Vectors
Gene Therapy in Skeletal Muscle Mediated by Adeno-Associated Virus Vectors
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DOI：
10.1007/978-1-61779-370-7_5
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Dickson, J. George
中科院分区：
文献类型：
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作者：
Qiao, Chunping;Koo, Taeyoung;Dickson, J. George
腺相关病毒（AAV）是肌肉定向基因治疗最有前途的基因递送载体。 AAV 对肌肉细胞的天然趋向性、长期持续的转基因表达、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 载体介导的基因传递可增强肌营养不良蛋白和肌聚糖等肌肉结构蛋白，为肌营养不良症患者带来了巨大的希望。此外，肌肉还可用作AAV载体的治疗平台，表达非肌肉分泌/调节途径蛋白，用于治疗糖尿病、动脉粥样硬化、血友病、癌症等。AAV载体可通过局部、区域和全身给药的方式递送至骨骼肌和心肌。成功的动物研究已经导致了一些涉及肌肉定向基因治疗的值得注意的临床试验。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mRNA表达的定量，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。
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.