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: 9765358
• 负责人: MATTHEW Scott ALEXANDER
• 金额: $ 31.81万
• 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/9765358
• 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; improved; in vivo; loss of function mutation; male; mdx mouse; member; 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.

项目摘要 杜氏肌营养不良症（DMD）是一种X连锁疾病，影响1：5000活男婴在美国每个 年DMD是由肌营养不良蛋白（DMD）基因的功能丧失突变引起的， 肌肉萎缩，12岁时失去肌肉，心律失常，成年早期因呼吸道疾病而死亡 失败虽然有几种新的策略（如外显子跳跃化合物，以创建部分功能性 肌营养不良蛋白）在临床试验中，他们都没有处理二级信号通路和随后的 DMD中发生的肌肉发病机制。越来越多的证据表明，DMD的遗传修饰剂具有 对患者DMD相关病理的结局和严重程度有显著影响。这些DMD基因 修饰剂具有作为治疗疾病的新的治疗切入点的潜力。先前 我的实验室发现miR-486是一种富含肌肉的microRNA，在DMD患者中表达减少。 骨骼肌活检。 在这个提议中，我们将检验miR-486及其靶点（DOCK 3）在细胞凋亡中发挥重要作用的假设。 骨骼肌的调节。我们有强有力的数据表明，miR-486的表达水平 减少和mRNA靶（DOCK 3）增加，对应于营养不良病理学的程度。在 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， 在正常和mdx 5cv背景下，miR-486敲除（KO）小鼠的肌肉性能和心脏重塑。 目的2着重于确定操纵miR-486和Dock 3表达对关键细胞的影响。 使用新型Dock 3条件性肌肉的下游信号通路，包括PTEN/AKT和Rac 1/RhoA 敲除小鼠。此外，我们将使用Ago 2测序（CLIP-seq）鉴定新的miR-486靶基因。 策略来鉴定miR-486的体内骨骼肌靶点。最后，Aim 3专注于使用瞬时miR-486 肌内递送的过表达策略（腺相关病毒载体和工程化外泌体） 并全身性地改善MDX 5CV小鼠在关键发育时间点的营养不良症状。我们 DMD患者肌肉细胞系、miR-486 KO和Dock 3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 我们严格评估使用microRNA（miR-486）过表达作为治疗糖尿病的新疗法。 DMD的治疗