Down-regulation of microRNA-206 in striated muscle of mdx mice improves muscle function.

 描述（由申请人提供）：杜氏肌营养不良症（DMD）是一种严重的肌肉萎缩性疾病，是全球最普遍的遗传性肌肉疾病。它是由肌营养不良蛋白基因突变引起的，该基因通常编码位于肌肉膜上的重要结构和信号蛋白。在DMD中，肌营养不良蛋白在体内不存在或以低水平存在，使肌肉容易受到损伤。DMD患者随着时间的推移会失去运动功能，并因呼吸或心力衰竭而过早死亡。目前的治疗仅仅是治标不治本。 旨在替换或纠正突变基因的基因治疗策略已显示出治疗DMD的前景。重组腺相关病毒（AAV）是流行的基因递送载体，这是由于它们的非致病性性质和建立长期和有效的基因转移的能力。尽管如此，恢复抗肌萎缩蛋白不能完全缓解运动缺陷和预防轻度活动后的疲劳。虽然DMD是由单个基因突变引起的，但涉及许多继发性疾病机制，如缺血，纤维化和异常再生。解决多种病理机制的策略可能会提供更大的益处。 microRNA（miRNAs或miRs）是抑制其靶基因的小的调节RNA分子。一种骨骼肌限制性miRNA，miR-206，在营养不良的肌肉中高度上调。虽然其在疾病发作后的功能尚不清楚，但多个miR-206靶标已显示出对DMD的治疗益处，如血管内皮生长因子A（VEGF-A）和utrophin。因此，miR-206的下调及其对纠正机制的不利影响提出了治疗DMD的新策略。本提案旨在表征携带针对miR-206的反义序列的重组AAV载体（AAV-anti-miR-206）的治疗功效，探索其对两种次要病理机制的影响。 功能性缺血是营养不良表型的主要贡献者，并加剧肌肉损伤。为了更好地理解AAV-抗miR-206对肌肉血管化的作用，第一个目的将研究其增加已证实的治疗靶点VEGF-A表达的能力。这些实验还将确定AAV-抗miR-206是否可以重现与VEGF-A治疗相关的血管生长、功能和完整性。 使用肌营养不良蛋白（一种肌营养不良蛋白）增加膜稳定性可以延缓DMD进展并改善运动功能。为了确定AAV-抗miR-206是否影响肌肉结构完整性和病理学，第二个目的将探索其对肌营养蛋白表达的影响。拟议的实验将确定这种治疗是否改善整体肌肉病理学并防止进一步损伤。这些目标将使我们更好地了解miR-206的功能，并探索一种新的、多功能的DMD治疗策略。

 DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease and the most prevalent inherited muscle disorder worldwide. It is caused by a mutation in the dystrophin gene, which normally encodes an important structural and signaling protein located at the muscle membrane. In DMD, the dystrophin protein is absent or present at low levels within the body, rendering the muscle susceptible to damage. DMD patients lose motor function over time and die prematurely from respiratory or cardiac failure. Current treatments are merely palliative and do not target the underlying cause. Gene therapy strategies aimed at replacing or correcting mutated genes have shown promise for DMD. Recombinant adeno-associated viruses (AAVs) are popular gene delivery vehicles due to their non-pathogenic nature and ability to establish long-term and efficient gene transfer. Still, restoring dystrophin fails to completely alleviate motor deficits and prevent fatigue after mild activity. While DMD is caused by a mutation in a single gene, many secondary disease mechanisms are involved, such as ischemia, fibrosis and abnormal regeneration. A strategy that addresses multiple pathological mechanisms may provide greater benefit. MicroRNAs (miRNAs or miRs) are small, regulatory RNA molecules that inhibit their target genes. A skeletal muscle-restricted miRNA, miR-206, is highly up-regulated in dystrophic muscle. While its function after disease onset is not well-understood, multiple miR-206 targets have shown therapeutic benefit for DMD, such as vascular endothelial growth factor A (VEGF-A) and utrophin. Down-regulation of miR-206 and its detrimental effect on corrective mechanisms thus presents a novel strategy for treating DMD. This proposal aims to characterize the therapeutic efficacy of a recombinant AAV vector carrying an antisense sequence against miR-206 (AAV-anti-miR-206), exploring its impact on two secondary pathological mechanisms. Functional ischemia is a major contributor to the dystrophic phenotype and exacerbates muscle damage. To better understand the role of AAV-anti-miR-206 on muscle vascularization, the first Aim will investigate its ability to increase the expression of proven therapeutic target VEGF-A. These experiments will also determine if AAV-anti-miR-206 can recapitulate blood vessel growth, function and integrity associated with VEGF-A treatment. Increasing membrane stability using utrophin, a dystrophin paralog, can delay DMD progression and improve motor function. To determine if AAV-anti-miR-206 influences muscle structural integrity and pathology, the second Aim will explore its effect on utrophin expression. The proposed experiments will determine if this treatment improves overall muscle pathology and prevents further damage. Together these Aims will provide a better understanding of miR-206 function and explore a novel, multifunctional therapeutic strategy for DMD.