Therapeutic targeting of miR-128-1 in Duchenne muscular dystrophy

项目摘要/摘要 Duchenne肌营养不良症(DMD)是一种X连锁遗传性神经肌肉疾病，具有 全球发病率为每3,500-5,000名活男婴中就有一名，使其成为最常见的 肌肉营养不良症。DMD是由Dstrophin基因突变引起的，导致 由于骨骼肌和心肌的丧失而导致的进行性肌肉萎缩障碍。这还为时尚早 致命性疾病，大多数男性在20岁或30岁时死于心脏或呼吸系统疾病S或S 并发症。因此，迫切需要新的治疗途径来治疗 DMD作为目前的治疗方法，疗效有限。调控的分子机制 营养不良蛋白缺失对下游的有害影响尚不清楚。我们注意到，表达式 在人类DMD患者的肌肉和循环中，miR-128-1的microRNA升高， 在小鼠和斑马鱼DMD模型的肌肉中。此外，miR-128-1基因组座位是 在英国生物库中，明显与握力不足和肺肌肉功能不良有关 (&gt；300,000人)。斑马鱼和小鼠DMD模型的初步研究 已发现使用锁定核酸(LNA)反义寡核苷酸(ASO)抑制miR-128-1 显著缓解DMD的表型，包括肌肉萎缩和运动不耐受。 此外，我们的初步研究表明，抑制miR-128-1在很大程度上挽救了 一组与骨骼线粒体健康和能量有关的关键基因的表达 动态平衡，伴随着体外和体内线粒体生物发生和功能的改善 活着。在这个应用中，我们提出了一些研究来检验miR-128-1代表一个 关键的疾病修饰物，通过调节Dystrophin丢失的有害影响来协调 一组对线粒体健康很重要的关键靶基因和可能的靶基因 对肌肉新陈代谢至关重要的物质。在第一个目标中，我们将调查miR-128-1的作用 在介导DMD线粒体异常中的作用并评估是否改善 MiR-128-1抑制和药物激活剂联合应用对线粒体功能的影响 线粒体功能协同改善mdx5Cv小鼠肌肉功能障碍 DMD模型和人类DMD患者来源的肌肉细胞。此外，我们还将 Mdx5Cv小鼠miR-128-1靶基因的转录鉴定 分析和评估人类保护。在第二个目标中，我们将评估治疗 用LNA ASOS抑制miR-128-1在小鼠mdx5Cv DMD模型中的疗效 小鼠对miR-128-1和肌肉靶向腺相关病毒的条件KO接近。 拟议研究的成功完成将确定miR-128-1是否真的可以 代表了DMD强有力的治疗靶点，并揭示了其下游机制 由此miR-128-1介导了DMD的病理过程。

Project Summary/Abstract Duchenne muscular dystrophy (DMD), an X-linked inherited neuromuscular disorder, has a worldwide incidence of one in ~3,500-5,000 live male births, making it the most common muscular dystrophy. DMD is caused by mutations in the dystrophin gene, resulting in a progressive muscle-wasting disorder due to loss of skeletal and cardiac muscle. It is an early lethal disease, and most afflicted males die in their 20’s or 30’s of cardiac or respiratory complications. There is thus an urgent need for novel therapeutic avenues for the treatment of DMD as the current treatments have only limited efficacy. The molecular mechanisms mediating deleterious effects downstream of dystrophin loss remain unclear. We note that the expression of the miR-128-1 microRNA is elevated in the muscle and circulation of human DMD patients, and in muscle of mouse and zebrafish DMD models. Moreover, the miR-128-1 genomic locus is markedly linked to weak grip strength and poor lung muscle function in the UK Biobank (>300,000 individuals). Our preliminary studies from the zebrafish and mouse models of DMD have found that inhibition of miR-128-1 using locked nucleic acid (LNA) antisense oligos (ASO) dramatically mitigates the DMD phenotypes, including muscle atrophy and exercise intolerance. Furthermore, our preliminary studies have revealed that miR-128-1 inhibition largely rescues the expression of a suite of key genes involved in skeletal mitochondrial health and energy homeostasis, accompanied by improved mitochondrial biogenesis and function in vitro and in vivo. In this application, we propose studies to test the hypothesis that miR-128-1 represents a crucial disease modifier that orchestrates the deleterious effects of dystrophin loss by regulating a set of key target genes that are important for mitochondrial health and putative target genes that are critical for muscle metabolism. In the first Aim, we will investigate what roles miR-128-1 play in mediating mitochondrial abnormalities in DMD and assess whether improving mitochondrial function by combining miR-128-1 inhibition and pharmacological activators of mitochondrial function can synergistically ameliorate muscle dysfunction in the mdx5cv mouse DMD model and in human DMD patient-derived muscle cells. In addition, we will comprehensively identify miR-128-1 target genes in mdx5cv mice by performing transcriptomic analysis and assess human conservation. In the second Aim, we will evaluate the therapeutic efficacy of miR-128-1 inhibition in the mouse mdx5cv DMD model using LNA ASOs, as well as conditional mouse KO of miR-128-1 and muscle-targeted adeno-associated virus approaches. Successful completion of the proposed studies will determine whether miR-128-1 may indeed represent a powerful therapeutic target in DMD, and reveal the downstream mechanism whereby miR-128-1 mediates the DMD pathologies.