Expressing full-length dystrophin with AAV

描述（由申请人提供）：肌营养不良蛋白基因突变导致杜氏肌营养不良（DMD）和贝克尔肌营养不良（BMD）。这些毁灭性的疾病无法治愈。有效的治疗依赖于治疗体内所有受影响的肌肉。腺相关病毒（AAV）可以有效地将治疗基因传递到全身肌肉。因此，用AAV基因疗法取代缺陷性肌营养不良蛋白基因具有很大的治疗前景。然而，AAV载体具有5 kb的包装能力。这不足以递送11.06kb全长肌营养不良蛋白编码序列。出于这个原因，目前的AAV基因治疗被迫使用大规模截短的迷你或微小肌营养不良蛋白基因。在本申请中，我们提出开发一种新的三- AAV系统来表达全长蛋白。三种AAV载体的组合能力为完整的肌营养不良蛋白编码序列提供了足够的空间。然而，不同AAV载体之间的随机重组将导致全长蛋白质的非常低效的重构。其中，我们最近开发了一系列新的方法来促进不同AAV载体之间的单向重组。在这个项目中，我们将把这些新技术应用于tri-AAV全长肌营养不良蛋白载体的开发。我们的具体目标是：（1）设计并构建全长抗肌萎缩蛋白tri-AV载体，（2）通过局部基因转移在抗肌萎缩蛋白缺陷型mdx小鼠中评估全长抗肌萎缩蛋白tri-AV载体。我们相信，这个高回报的翻译项目将可能导致在未来的腺相关病毒介导的DMD基因治疗的突破。杜氏肌营养不良症（DMD）是一种威胁生命的疾病，影响相当大的人口（超过3,500名新生儿中的一名）。它是由肌营养不良蛋白基因突变引起的。最好的治疗方法是用正常基因替换突变基因。AAV载体可以表达肌营养不良蛋白基因的截短形式，但它不能表达全长蛋白。在这个翻译项目中，我们建议开发一种新的三-AAV系统来表达全长肌营养不良蛋白。如果成功，该项目将导致DMD基因治疗的重大突破。

DESCRIPTION (provided by applicant): Mutations in the dystrophin gene result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). There is no cure for these devastating diseases. An effective therapy relies on treating all affected muscles in the body. Adeno-associated virus (AAV) can efficiently deliver a therapeutic gene to all body muscles. Therefore, replacing the defective dystrophin gene with AAV gene therapy holds great therapeutic promise. However, AAV vector has a 5 kb packaging capacity. This is insufficient to deliver the 11.06 kb full-length dystrophin coding sequence. For this reason, current AAV gene therapy is forced to use the massively truncated mini- or micro-dystrophin genes. In this application, we propose to develop a novel tri- AAV system to express the full-length protein. The combined capacity of three AAV vectors provides enough room for the full dystrophin coding sequence. However, random recombination among different AAV vectors will lead to very inefficient reconstitution of the full-length protein. We, among others, have recently developed a series of novel approaches to facilitate unidirectional recombination between different AAV vectors. In this project, we will apply these new technologies to the development of the tri-AAV full-length dystrophin vectors. Our specific aims are: (1) to design and construct full-length dystrophin tri-AAV vectors, and (2) to evaluate the full-length dystrophin tri-AAV vectors in dystrophin-deficient mdx mice by local gene transfer. We believe that this high payback translational project will likely lead to a breakthrough in AAV-mediated DMD gene therapy in the future. Duchenne muscular dystrophy (DMD) is a life threatening disease affecting a fairly large population (more than one in 3,500 newborns). It is caused by dystrophin gene mutations. The best therapy is to replace the mutated gene with a normal gene. AAV vector can express truncated versions of the dystrophin gene but it cannot express the full-length protein. In this translational project, we propose to develop a novel tri-AAV system to express the full-length dystrophin protein. If successful, this project will lead to a big breakthrough in DMD gene therapy.