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

 描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种严重的肌肉萎缩疾病，也是全世界最常见的遗传性肌肉疾病。它是由肌营养不良蛋白基因的突变引起的，该基因通常编码位于肌膜上的一种重要的结构和信号蛋白。在DMD中，营养不良蛋白在体内不存在或存在于低水平，使肌肉容易受到损害。随着时间的推移，DMD患者会失去运动功能，并因呼吸或心脏衰竭而过早死亡。目前的治疗只是治标不治本，并不针对根本原因。旨在替换或纠正突变基因的基因治疗策略已经显示出对DMD的希望。重组腺相关病毒(AAV)由于其非致病性和建立长期有效的基因转移的能力，是一种受欢迎的基因传递工具。尽管如此，恢复营养不良蛋白并不能完全缓解运动障碍，并防止轻度运动后的疲劳。虽然DMD是由单个基因的突变引起的，但也涉及许多继发性疾病机制，如缺血、纤维化和异常再生。一种应对多种病理机制的策略可能会带来更大的好处。MicroRNAs(miRNAs或miRs)是一种小的、调节的RNA分子，可以抑制其目标基因。一种骨骼肌限制性miRNA miR-206在营养不良肌肉中高度上调。虽然miR-206在疾病发生后的作用尚不清楚，但多个miR-206靶点已显示出对DMD的治疗益处，如血管内皮生长因子A(VEGF-A)和中性粒细胞营养素。因此，miR-206的下调及其对纠正机制的不利影响为治疗DMD提供了一种新的策略。本研究旨在鉴定携带miR-206反义序列的重组AAV载体(AAV-anti-miR-206)的治疗效果，探讨其对两种继发性病理机制的影响。功能性缺血是营养不良表型的主要因素，并加重肌肉损伤。为了更好地了解AAV-anti-miR-206在肌肉血管形成中的作用，第一个目的是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-anti-miR-206是否可以概括与血管内皮生长因子-A治疗相关的血管生长、功能和完整性。使用营养不良蛋白(Dystrophin Paralog)来增加膜的稳定性可以延缓DMD的进展并改善运动功能。为了确定AAV-anti-miR-206是否影响肌肉结构完整性和病理学，第二个目的将探讨其对utroin表达的影响。拟议中的实验将确定这种治疗是否改善整体肌肉病理并防止进一步损害。这些目标将有助于更好地了解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.