Genetic and pharmacologic elimination of myotonia from myotonic dystrophy type 1

通过遗传和药物消除 1 型强直性肌营养不良引起的肌强直

• 批准号: 10750357
• 负责人: Matthew Thomas Sipple
• 金额: $ 4.77万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750357
• 关键词: 3' Untranslated Regions; Abate; Address; Adult; Affect; Alternative Splicing; Anti-Arrhythmia Agents; Antiepileptic Agents; Automobile Driving; Back; Binding Proteins; Biological Assay; CUG repeat; Cardiac; Cataract; Characteristics; Chloride Channels; Chlorides; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Disease; Disease Progression; Distal; Embryo; Excision; Exons; Family; Fiber; Functional disorder; Genes; Genetic; Genetic Transcription; Goals; Hand Strength; Histologic; Histology; Histopathology; In Vitro; Insulin Resistance; Intervention; Ion Channel; Knock-out; Limb structure; Link; Measurement; Messenger RNA; Methods; Modeling; Mus; Muscle; Muscle Weakness; Muscular Dystrophies; Myopathy; Myotonia; Myotonic Dystrophy; Myotonic dystrophy type 1; Neuromuscular Junction; Nonsense-Mediated Decay; Oropharyngeal; Outcome; Pathogenesis; Pathology; Patients; Pattern; Pharmaceutical Preparations; Pharmacological Treatment; Phenotype; Physiological; Play; Protein Kinase; Quality of life; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Resistance; Role; Running; Severities; Skeletal Muscle; Sodium Channel Blockers; Stimulus; Structure; Symptoms; System; Terminator Codon; Therapeutic; Transcript; Translating; Trinucleotide Repeat Expansion; Trinucleotide Repeats; autosome; clinically relevant; disease phenotype; experimental study; improved; in vivo; mRNA Decay; mouse model; muscle physiology; novel; pharmacologic; premature; prevent; ranolazine; transcriptome sequencing; treadmill; treatment effect; voltage; wasting

Project Summary: Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy that features prominent muscular symptoms, including myotonia, weakness, and wasting, as well as a diverse array of extra-muscular manifestations, including ocular cataracts, cardiac conduction abnormalities, and insulin resistance among others. DM1 is an autosomal dominant disorder that has been linked to the CTG trinucleotide repeat (TNR) expansion in the 3' UTR of the Dystrophia Myotonica Protein Kinase (DMPK) gene. DM1 patients possess greater than 50 (CTG) repeats and transcribe toxic RNA products capable of sequestering RNA-binding proteins, such as the Muscleblind-like (MBNL) family of splicing factors. This alters the available pool of splicing factors leading to changes in the alternative splicing for more than 100 developmentally regulated transcripts. Overall, these changes in splicing display a reversion back to embryonic patterns, with one example being the splicing of transcripts for ClC-1, a voltage-gated chloride channel expressed in skeletal muscle. In DM1, there is increased inclusion of exon 7a in Clcn1 transcripts, which causes a frameshift, premature termination codon, and a non-functional ion channel. This yields decreased inhibitory chloride conductance that has been directly linked to muscle hyperexcitability causing myotonia. In patients with DM1, the distribution of myotonia tends to correlate with other muscular symptoms—including weakness and wasting—with the highest severity of these symptoms localized to muscles of the distal extremities and oropharynx. This combined with evidence that approximately one-fourth of the expression level changes of transcripts in myofibers could be due to myotonia and not toxic-RNA has led to the hypothesis that myotonia plays a central role in driving the other skeletal muscle pathology in DM1. To investigate this, we developed a novel mouse line (ClC-1∆E7a/∆E7a) resistant to myotonia caused by the aberrant inclusion of E7a to eliminate myotonia from models of DM1. We will cross this novel line with the Mbnl1-/- and Mbnl1-/-/Mbnl2-/+ mouse models of DM1 to eliminate myotonia from these models and compare the changes in muscle physiology both in vitro (e.g., force contraction) and in vivo (e.g., grip strength), histopathology (e.g., central nucleation, fiber-type distribution), and genetic regulation (RNAseq) between myotonic and non-myotonic progeny. Thus far, we have successfully eliminated myotonia in Mbnl1-/-/ClC-1∆E7a mice and have observed significant histological and splicing differences compared to myotonic Mbnl1-/-/ClC-1+/+ mice, with the non-myotonic mice featuring phenotypes closer to wild-type. Then, we will replicate these studies with long-term anti-myotonic treatment of DM1 model mice with ranolazine with the goal that this could represent a myo-protective approach to aid DM1 patients.

项目总结： 强直性肌营养不良1型(DM1)是最常见的成人型肌营养不良症，其显著特点是 肌肉症状，包括肌强直、虚弱和消瘦，以及一系列不同的肌肉外 表现包括眼部白内障、心脏传导异常和胰岛素抵抗 其他。DM1是一种常染色体显性遗传病，与CTG三核苷酸重复(TNR)相关 Dystrophia Myotonica蛋白激酶(DMPK)基因3‘端非编码区的扩增DM1患者拥有 大于50(CTG)重复并转录能够隔离RNA结合蛋白的有毒RNA产物， 例如粘虫样(MBNL)家族的剪接因子。这改变了可用的剪接因子池 导致100多个发育调节转录本的选择性剪接发生变化。总的来说， 剪接的这些变化显示了向胚胎模式的回归，剪接就是一个例子 ClC-1，一种在骨骼肌中表达的电压门控氯通道的转录本。在DM1中，有 Clcn1转录本中增加了外显子7a的包含，这导致了移码、提前终止密码子， 和不起作用的离子通道。这会降低直接产生的抑制氯离子电导。 与导致肌强直的肌肉过度兴奋有关。在DM1患者中，肌强直的分布趋向于 与其他肌肉症状相关--包括虚弱和消瘦--这些症状的严重性最高 症状局限于四肢远端和口咽部的肌肉。再加上有证据表明 大约四分之一的转录本在肌纤维中的表达水平变化可能是由于肌强直 而且没有毒性--RNA导致了一种假说，即肌强直在驱动其他骨骼肌的过程中起着核心作用 DM1中的病理学。为了研究这一点，我们开发了一种新的抗肌强直小鼠品系(clc-1∆E7a/∆E7a) 由异常包含E7a以消除DM1模型中的肌强直所致。我们将越过这条新奇的界线 用DM1的MBNL1-/-和MBNL1-/-/Mbnl2-/+小鼠模型消除这些模型中的肌强直 比较体外(例如，力量收缩)和体内(例如，握力)的肌肉生理学变化， 组织病理学(例如，中央成核、纤维类型分布)和基因调控(RNAseq) 强直性和非强直性子代。到目前为止，我们已经成功地消除了MBNL1-/-/Clc-1∆E7a的肌强直 并观察到与强直性肌强直MBNL1-/-/ClC-1+/+相比，在组织学和剪接方面存在显著差异 小鼠，非强直性肌强直小鼠的表型更接近野生型。然后，我们将重复这些研究 用雷诺嗪对DM1模型小鼠进行长期抗肌张力治疗，目的是这可能代表 一种帮助DM1患者的心肌保护方法。