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.

项目概要： 强直性肌营养不良 1 型 (DM1) 是最常见的成人发病性肌营养不良，其特点是 肌肉症状，包括肌强直、虚弱和消瘦，以及各种肌肉外症状 症状，包括眼部白内障、心脏传导异常和胰岛素抵抗 DM1 是一种常染色体显性遗传疾病，与 CTG 三核苷酸重复序列 (TNR) 有关。 DM1 患者的肌强直营养不良蛋白激酶 (DMPK) 基因 3' UTR 发生扩增。 超过 50 个 (CTG) 重复序列并转录能够隔离 RNA 结合蛋白的有毒 RNA 产物， 例如肌盲样 (MBNL) 剪接因子家族，这会改变可用的剪接因子库。 导致 100 多个发育调控转录本的选择性剪接发生变化。 剪接的这些变化显示出对胚胎模式的回归，其中一个例子是剪接 ClC-1 的转录本，一种在骨骼肌中表达的电压门控氯离子通道。 Clcn1 转录本中外显子 7a 的含量增加，这会导致移码、过早终止密码子， 和非功能性离子通道，这会直接降低抑制性氯电导。 与引起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（包括虚弱和消瘦）相关，这些症状的严重程度最高 症状局限于四肢远端和口咽部的肌肉，这与证据相结合。 肌纤维转录物表达水平变化的大约四分之一可能是由于肌强直引起的 而不是有毒的RNA导致了这样的假设：肌强直在驱动其他骨骼肌方面发挥着核心作用 为了研究这一点，我们开发了一种新型的抗肌强直小鼠品系 (ClC-1ΔE7a/ΔE7a)。 为了消除 DM1 模型中的肌强直，我们将跨越这条新线。 与 DM1 的 Mbnl1-/- 和 Mbnl1-/-/Mbnl2-/+ 小鼠模型一起消除这些模型中的肌强直， 比较体外（例如力收缩）和体内（例如握力）肌肉生理学的变化， 组织病理学（例如，中心成核、纤维类型分布）和遗传调控 (RNAseq) 到目前为止，我们已经成功消除了 Mbnl1-/-/ClC-1ΔE7a 的肌强直和非肌强直后代。 小鼠并观察到与强直性 Mbnl1-/-/ClC-1+/+ 相比显着的组织学和剪接差异 小鼠，非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强直治疗，目的是这可以代表 一种帮助 DM1 患者的肌肉保护方法。