Genetic and pharmacologic elimination of myotonia from myotonic dystrophy type 1

项目摘要： 强直性肌营养不良1型（DM 1）是最常见的成人发病的肌营养不良症，其特征在于突出的 肌肉症状，包括肌强直，虚弱和消瘦，以及各种各样的肌外 临床表现包括白内障、心脏传导异常和胰岛素抵抗， 他人DM 1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。 在肌强直性肌营养不良症蛋白激酶（DMPK）基因的3' UTR中的扩增。DM 1患者具有 大于50个（CTG）重复并转录能够螯合RNA结合蛋白的毒性RNA产物， 例如肌盲样（MBNL）剪接因子家族。这改变了剪接因子的可用库 导致超过100种发育调节转录物的可变剪接发生变化。总体而言， 剪接的这些变化显示了向胚胎模式的逆转，其中一个例子是 ClC-1是一种在骨骼肌中表达的电压门控氯离子通道。在DM 1中， 在Clcn 1转录物中增加外显子7a的包含，这导致移码，提前终止密码子， 和非功能性离子通道。这导致抑制性氯离子电导降低， 与引起肌强直的肌肉过度兴奋有关在DM 1患者中，肌强直的分布倾向于 与其他肌肉疾病相关-包括虚弱和消瘦-这些疾病的严重程度最高 症状局限于四肢远端和口咽部的肌肉。这与证据相结合， 肌纤维中转录物表达水平的变化约有四分之一可能是由于肌强直 而不是有毒的RNA导致了这样的假设，即肌强直在驱动其他骨骼肌中起着核心作用， DM 1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的小鼠品系（ClC-1 E7 a/E7 a 由E7 a的异常包含引起，以消除DM 1模型中的肌强直。我们将跨越这条新的界线 用DM 1的Mbnl 1-/-和Mbnl 1-/-/Mbnl 2-/+小鼠模型治疗，以从这些模型中消除肌强直， 比较体外肌肉生理学的变化（例如，力收缩）和体内（例如，握力）， 组织病理学（例如，中心成核，纤维型分布），和遗传调控（RNAseq）之间 肌强直和非肌强直后代。到目前为止，我们已经成功地消除了Mbnl 1-/-/ClC-1 β E7 a中的肌强直， 与肌强直性Mbnl 1-/-/ClC-1+/+相比， 小鼠，非肌强直小鼠的表型更接近野生型。然后，我们将复制这些研究 用雷诺嗪对DM 1模型小鼠进行长期抗肌强直治疗，目的是代表 一种帮助1型糖尿病患者的肌肉保护方法。

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.