Gene Therapy for a severe DMD Animal Model

描述（由申请人提供）：杜氏肌营养不良症（DMD），由肌营养不良蛋白基因突变引起，是最常见的致残性和致命性肌肉疾病，每3500名男性中就有一名患有DMD。最近，我们已经产生了一系列高度截短的迷你肌营养不良蛋白基因，在“非必需”区域，包括中央杆结构域的一部分和非常C-末端结构域有大的缺失。这些小基因足够小，可以包装到腺相关病毒（AAV）载体和足够大，以保持高功能，当在mdx小鼠局部肌肉内基因递送后进行测试。然而，mdx小鼠虽然是一种常用的DMD动物模型，但离理想的DMD动物模型还很远。虽然表现出许多与人类患者相似的症状，例如肌肉病理学，但mdx小鼠并没有像人类患者那样寿命缩短，也没有表现出整体肌肉无力和骨骼痉挛。这种现象是由于utrophin基因（一种抗肌萎缩蛋白类似物）的上调，部分补偿了mdx小鼠中抗肌萎缩蛋白的缺陷。通过敲除肌营养不良蛋白和utrophin基因（双KO），两个团队最近开发了一种严重的DMD小鼠模型，该模型密切反映了人类患者中观察到的每一种主要缺陷，包括寿命缩短，严重的肌肉无力和骨骼挛缩，为新疗法的更严格测试提供了更真实的小动物模型。在这项资助提案中，我们将使用新的双KO小鼠来大力测试新的mini-dystrophin基因是否能够局部和全身地拯救肌肉功能，更重要的是，改善整体健康并延长严重DMD动物的寿命，这是开发临床有效基因治疗策略的关键。本课题的研究内容包括：1）利用转基因小鼠技术，研究mini-dystrophin基因在双基因敲除小鼠中的生物学/治疗功能; 2）局部肌肉注射AAV载体后，mini-dystrophin基因在幼年和成年双基因敲除小鼠中的治疗效果; 3）全身性基因递送及其在大肌肉群和全身的治疗效果; 4）可与小基因一起提供协同效应沿着以有益于营养不良肌肉的替代治疗基因。

DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common, disabling and lethal muscle disease, afflicting one of every 3500 males. Recently, we have generated a series of highly truncated mini-dystrophin genes that had large deletions in the "non-essential" regions including part of the central rod domain and the very C-terminus domain. These minigenes were small enough to be packaged into adeno-associated virus (AAV) vectors and large enough to preserve high functionality, when tested in mdx mice after local intramuscular gene delivery. However, the mdx mice are far from an ideal DMD animal model although it is a commonly used one. While manifesting many similar symptoms of the human patients such as the muscle pathology, the mdx mice do not suffer shortened lifespan and do not show overall muscle weakness and skeletal contractures as do the human patients. This phenomenon is due to the up-regulation of utrophin gene (a dystrophin analogue) that partially compensates the defects of dystrophin in the mdx mice. By knocking out both dystrophin and utrophin genes (double-KO), two teams have recently developed a severe DMD mouse model that closely reflects every major deficiency seen in the human patients including much shortened life-span, severe muscle weakness and skeletal contractures, offering a more truthful small animal model for more stringent tests of new therapeutics. In this grant proposal, we will use the newly available double-KO mice to vigorously test the hypothesis whether the novel mini-dystrophin genes are able to rescue the muscle functions locally and systemically, and more importantly, to improve the overall health and prolong the life-span of the severe DMD animal, which is key to the development of a clinically efficacious gene therapy strategy. In this proposal, we will investigate 1) biological/therapeutic functions of mini-dystrophin genes in the double-KO mice using the transgenic mouse technology; 2) therapeutic effects of mini-dystrophin genes in both young and adult double-KO mice after local intramuscular injection of AAV vectors; 3) systemic gene delivery and its therapeutic effects in large groups of muscles and the entire body; 4) alternative therapeutic genes that may offer synergistic effects along with the minigenes to benefit the dystrophic muscles.