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

项目总结/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， 全世界范围内的发病率约为3，500 - 5，000例活产男婴中的1例，使其成为最常见的 肌肉萎缩症DMD是由肌营养不良蛋白基因突变引起的， 由于骨骼肌和心肌的损失而导致的进行性肌肉萎缩。性而种的苹果是早熟 致命的疾病，大多数患病的男性在20或30岁时死于心脏或呼吸系统疾病。 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。分子机制介导 抗肌萎缩蛋白损失下游的有害作用仍不清楚。我们注意到， miR-128-1 microRNA在人DMD患者的肌肉和循环中升高， 在小鼠和斑马鱼DMD模型的肌肉中。此外，miR-128-1基因组位点是 在英国生物样本库中， （超过300 000人）。我们对斑马鱼和小鼠DMD模型的初步研究 已经发现使用锁核酸（LNA）反义寡核苷酸（阿索）抑制miR-128-1， 显著减轻DMD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，miR-128-1抑制在很大程度上挽救了 一组参与骨骼线粒体健康和能量的关键基因的表达 体内平衡，伴随着体外和体内线粒体生物发生和功能的改善， vivo.在本申请中，我们提出了研究来检验miR-128-1代表一种新的表达的假设。 一种重要的疾病修饰因子，通过调节抗肌萎缩蛋白丢失的有害作用， 一组对线粒体健康重要的关键靶基因和推定的靶基因 对肌肉新陈代谢至关重要。在第一个目标中，我们将研究miR-128-1在细胞内的作用， 在介导DMD线粒体异常中发挥作用，并评估是否改善 通过结合miR-128-1抑制和线粒体功能的药理学激活剂， 线粒体功能可以协同改善mdx 5cv小鼠的肌肉功能障碍 DMD模型和人DMD患者来源的肌细胞。此外，我们将 通过转录组学方法全面鉴定mdx 5cv小鼠中的miR-128-1靶基因， 分析和评估人类保护。在第二个目标中，我们将评估治疗 使用LNA ASO的小鼠mdx 5cv DMD模型中miR-128-1抑制的功效，以及 miR-128-1的条件性小鼠KO和肌肉靶向腺相关病毒方法。 成功完成拟议的研究将确定miR-128-1是否真的可能 代表DMD的一个强有力的治疗靶点，并揭示下游机制 由此miR-128-1介导DMD病理。

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.