Gene Therapy for a severe DMD Animal Model

描述（由申请人提供）：由肌营养不良蛋白基因突变引起的杜氏肌营养不良症（DMD）是最常见、致残和致命的肌肉疾病，每3500名男性中就有一人患有此病。最近，我们已经生成了一系列高度截断的迷你肌营养不良蛋白基因，这些基因在“非必需”区域有大量缺失，包括部分中央杆结构域和非常c端结构域。这些小基因足够小，可以被包装成腺相关病毒（AAV）载体，并且足够大，可以在mdx小鼠中进行局部肌肉注射基因递送后保持高功能。然而，mdx小鼠虽然是一种常用的DMD动物模型，但距离理想的DMD动物模型还很远。虽然mdx小鼠表现出许多与人类患者相似的症状，如肌肉病理，但它们的寿命没有缩短，也没有像人类患者那样表现出整体肌肉无力和骨骼挛缩。这种现象是由于在mdx小鼠中，肌营养不良蛋白基因（一种肌营养不良蛋白类似物）的上调部分补偿了肌营养不良蛋白的缺陷。通过敲除肌营养不良蛋白和肌营养不良蛋白基因（double-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的每一个主要缺陷，包括寿命大大缩短、严重肌肉无力和骨骼挛缩，为更严格的新疗法测试提供了一个更真实的小动物模型。在本次拨款申请中，我们将利用新获得的双ko小鼠，大力验证新型mini-dystrophin基因是否能够局部和系统地挽救肌肉功能，更重要的是，是否能够改善重度DMD动物的整体健康状况和延长寿命，这是开发临床有效的基因治疗策略的关键。在本研究中，我们将利用转基因小鼠技术研究微肌营养不良蛋白基因在双ko小鼠中的生物学/治疗功能；2)局部肌内注射AAV载体后，小肌营养不良蛋白基因对幼龄和成年双ko小鼠的治疗作用；3)全身性基因传递及其在大群肌肉和全身的治疗效果；4)替代治疗基因，可能提供协同效应与miniigenes，以受益于营养不良的肌肉。

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.