Gene Therapy for a severe DMD Animal Model

描述(申请人提供)：Duchenne肌营养不良症(DMD)，由dystrophin基因突变引起，是最常见的致残性和致命性肌肉疾病，每3500名男性中就有一人患有此病。最近，我们已经产生了一系列高度截断的迷你肌营养不良蛋白基因，这些基因在包括部分中央杆状结构域和非常C-末端结构域的“非必要”区域有较大的缺失。这些小基因足够小，可以包装成腺相关病毒(AAV)载体，也足够大，当在局部肌肉内基因传递后在MDX小鼠身上进行测试时，足够大，以保持高功能。然而，尽管MDX小鼠是一种常用的DMD动物模型，但它远不是理想的DMD动物模型。虽然表现出许多与人类患者相似的症状，如肌肉病理，但MDX小鼠没有遭受寿命缩短的痛苦，也没有像人类患者那样表现出全面的肌肉无力和骨骼痉挛。这种现象是由于滋养蛋白基因(一种抗肌营养不良蛋白类似物)上调，部分弥补了MDX小鼠抗肌营养不良蛋白的缺陷。通过敲除营养不良蛋白和促性腺激素基因(Double-KO)，两个团队最近开发出了一种严重的DMD小鼠模型，该模型几乎反映了人类患者的所有主要缺陷，包括寿命大大缩短、严重的肌肉无力和骨骼痉挛，为更严格的新疗法测试提供了更真实的小动物模型。在这项拨款建议中，我们将利用新获得的双KO小鼠来积极检验新的微型Dstrophin基因是否能够局部和系统地挽救肌肉功能，更重要的是，改善重症DMD动物的整体健康和延长其寿命，这是开发临床有效的基因治疗策略的关键。在这个建议中，我们将1)利用转基因小鼠技术研究微型dystrophin基因在双KO小鼠中的生物/治疗功能；2)局部肌肉注射AAV载体后微型dystrophin基因在幼年和成年双KO小鼠中的治疗作用；3)全身性基因的传递及其在大群肌肉和全身的治疗作用；4)替代治疗基因，其与微型基因有协同作用，使营养不良的肌肉受益。

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.