A MIR-486/DOCK3 SIGNALING AXIS MODULATES DYSTROPHIN-DEFICIENT PATHOLOGY

MIR-486/DOCK3 信号轴调节肌营养不良蛋白缺乏的病理学

• 批准号: 10171875
• 负责人: MATTHEW Scott ALEXANDER
• 金额: $ 32.88万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-17 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171875
• 关键词: Affect; Age; Apoptosis; Arrhythmia; Base Pairing; Base Sequence; Binding; Biological Markers; Biopsy; Birth; Cardiac; Cell Death; Cell Line; Cell fusion; Cells; Cessation of life; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Data; Development; Disease; Disease Progression; Drug Compounding; Duchenne muscular dystrophy; Dystrophin; Evaluation; Exercise; Gene Expression; Genes; Genetic; Histology; Human; Impairment; Intramuscular; Knock-out; Knockout Mice; Laboratories; Link; Mediating; Membrane; Membrane Proteins; Messenger RNA; Methods; MicroRNAs; Molecular; Mus; Muscle; Muscle Cells; Muscle function; Muscular Atrophy; Myocardium; Myopathy; Outcome; PTEN gene; PTEN protein; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Performance; Phenotype; Play; Protein Family; Proteins; Proto-Oncogene Proteins c-akt; RHOA gene; Regulation; Respiratory Failure; Role; Seeds; Serum; Severities; Severity of illness; Signal Pathway; Signal Transduction; Site; Skeletal Muscle; Stress; Symptoms; Testing; Therapeutic; Time; Viral; adeno-associated viral vector; boys; combinatorial; crosslinking and immunoprecipitation sequencing; emerging adult; engineered exosomes; exon skipping; exosome; experience; heart function; human disease; human model; improved; in vivo; loss of function mutation; male; mdx mouse; member; miRNA expression profiling; mouse model; notch protein; novel; novel strategies; novel therapeutics; overexpression; predictive marker; respiratory; skeletal; skeletal muscle wasting

PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is an X-linked disorder that affects 1:5000 live male births in the US each year. DMD is caused by loss-of-function mutations in the DYSTROPHIN (DMD) gene that results in severe muscle wasting, loss of ambulation by age 12, cardiac arrhythmia, and death in early adulthood due to respiratory failure. While there are several novel strategies (such as exon-skipping compounds to create partially-functional Dystrophin protein) in clinical trials, none of them deal with the secondary signaling pathways and subsequent muscle pathogenesis that occurs in DMD. There is growing evidence that genetic modifiers of DMD have a significant impact on the outcomes and severity of DMD-related pathologies in patients. These DMD genetic modifiers have the potential to serve as novel therapeutic entry points for the treatment of diseases. Previously my laboratory identified miR-486 as a muscle-enriched microRNA that is decreased in expression in DMD patient skeletal muscle biopsies. In this proposal, we will test the hypothesis that miR-486 and its target (DOCK3) play a significant role in the regulation of skeletal muscle. We have strong data that demonstrates that miR-486 expression levels decrease and an mRNA target (DOCK3) increases corresponding with the degree of dystrophic pathology. In Aim 1, we will characterize the functional role of miR-486 on skeletal and heart muscles via characterizing muscle performance and cardiac remodeling in miR-486 knockout (KO) mice on normal and mdx5cv backgrounds. Aim 2 focuses on identifying the consequences of manipulating miR-486 and Dock3 expression on key downstream signaling pathways including PTEN/AKT and Rac1/RhoA using a novel Dock3 conditional muscle knockout mouse. Additionally, we will identify novel miR-486 target genes using Ago2-sequencing (CLIP-seq) strategy to identify in vivo skeletal muscle targets of miR-486. Lastly, Aim 3 focuses on using transient miR-486 overexpression strategies (Adeno-associated viral vectors and engineered exosomes) delivered intramuscularly and systemically to improve dystrophic symptoms at key developmental time points in mdx5cv mice. Our combinatorial use of DMD patient muscle cell lines, miR-486 KO and Dock3 conditional muscle KO mice, and experience evaluating genetic and drug compounds in mouse models of human diseases will allow us to rigorously evaluate the use of a microRNA (miR-486) overexpression as a novel therapy for the treatment of DMD.

项目总结 Duchenne肌营养不良症(DMD)是一种X连锁疾病，在美国，每5000名活男婴中就有1人受到影响 年。DMD是由营养不良蛋白(DMD)基因功能丧失突变引起的，这种突变导致严重的 肌肉萎缩，12岁时失去行走能力，心律失常，以及成年早期死于呼吸系统 失败了。虽然有几种新的策略(例如，跳过外显子的化合物来创建部分功能 在临床试验中，它们都没有涉及二级信号通路和随后的 发生在DMD中的肌肉发病机制。越来越多的证据表明，DMD的遗传修饰物具有 对患者DMD相关病理的转归和严重程度有显著影响。这些DMD基因 修饰剂有可能成为治疗疾病的新的治疗切入点。先前 我的实验室确认miR-486是一种肌肉丰富的microRNA，在DMD患者中表达降低 骨骼肌活组织检查。 在这个提案中，我们将检验miR-486及其靶基因(DOCK3)在 对骨骼肌的调节。我们有强有力的数据表明miR-486的表达水平 随着营养不良病理程度的加重，靶基因(DOCK3)的表达增加。在……里面 目的1，我们将通过表征miR-486在骨骼肌和心肌中的功能作用来表征miR-486的功能 在正常和mdx5Cv背景下miR-486基因敲除(KO)小鼠的肌肉表现和心脏重构。 目标2侧重于确定操纵key上的miR-486和Dock3表达式的后果 使用新的Dock3条件肌肉的包括PTEN/AKT和rac1/RhoA在内的下游信号通路 基因敲除老鼠。此外，我们还将使用ago2测序技术(lip-seq)鉴定新的miR-486靶基因。 确定miR-486体内骨骼肌靶向的策略。最后，目标3侧重于使用瞬变miR-486 肌肉注射过表达策略(腺相关病毒载体和工程外切体) 系统地改善mdx5Cv小鼠关键发育时间点的营养不良症状。我们的 DMD患者肌肉细胞系、miR-486KO和Dock3条件性肌肉KO小鼠的组合使用， 在人类疾病的小鼠模型中评估遗传和药物化合物的经验将允许 美国将严格评估使用microRNA(miR-486)过度表达作为一种新的治疗方法 DMD的治疗。