Therapeutic approach targeting RNA disease in myotonic dystrophy

描述（由申请人提供）：1型肌强直性营养不良（DM1）是最常见的肌肉营养不良形式，与进行性残疾和过早死亡有关。目前还没有治疗方法可以逆转DM1患者的肌肉萎缩和无力，或者减缓疾病的进展。这种疾病是由DMPK基因3'非翻译区CTG重复序列的扩增引起的。最近的研究结果表明，DM1涉及一种新的rna介导的疾病机制，由突变的DMPK mRNA启动，独立于DMPK蛋白。突变等位基因的转录本包含一个扩展的CUG重复序列，突变RNA的致病作用显然来自这个多聚（CUG）exp元件。例如，我们发现在完全不同的mRNA的3'非翻译区表达poly(CUG)exp可以在转基因小鼠中重现疾病。我们的合作研究已经建立了DM1发病机制的多步骤模型：(1)突变等位基因的转录产生poly(CUG)exp RNA；(2)突变转录本在细胞核中以离散灶（核糖核灶）积累；(3)肌盲（MBNL）家族中的剪接因子，主要是MBNL1，被隔离在核糖核病灶中；(4) MBNL1蛋白活性降低导致一组前mrna的选择性剪接异常调节，如肌上皮氯离子通道，ClC-1；(5)发育不适当的剪接异构体的表达导致DM1症状，如肌强直。为了支持该模型，我们发现MBNL1蛋白被广泛地募集到核糖核灶中，以至于在核质的其他地方明显减少，并且MBNL1敲除小鼠中的剪接缺陷或剪接病与DM1患者中观察到的非常相似。该模型预测，DM1在特定细胞核中的作用取决于与MBNL1蛋白供应相关的多聚（CUG）exp积累水平，减少多聚（CUG）exp RNA积累或抑制其与MBNL1相互作用的治疗有可能阻止疾病进展，甚至实现表型逆转。本提案的目的是开发检测方法来筛选以下化合物：(1)逆转DM1细胞模型中的剪接病变；(2)抑制细胞中MBNL1蛋白对poly(CUG)exp RNA的识别；(3)在体外抑制poly(CUG)exp RNA与MBNL1蛋白的相互作用。拟议的研究将在DM1的实验治疗作为其主要焦点的环境中进行。因此，筛选项目的新发现可以在适当的模型系统中迅速进行临床前测试，并转化为治疗试验。肌强直性营养不良是成人中最常见的肌肉营养不良形式，可导致进行性残疾和过早死亡。目前还没有治疗方法可以改善肌强直性营养不良患者的肌肉无力，或减缓疾病的进展。该项目的目标是发现可以开发成有效治疗肌强直性营养不良的药物。

DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1), the most prevalent form of muscular dystrophy, is associated with progressive disability and premature death. Presently there are no treatments that reverse the muscle wasting and weakness in patients with DM1, or that slow the disease progression. This disorder is caused by an expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Recent findings indicate that DM1 involves a novel RNA-mediated disease mechanism, initiated by the mutant DMPK mRNA, independently of DMPK protein. The transcripts from the mutant allele contain an expanded CUG repeat, and the pathogenic effects of the mutant RNA clearly derive from this poly(CUG)exp element. For example, we found that expression of poly(CUG)exp in the 3' untranslated region of an entirely different mRNA can recapitulate the disease in transgenic mice. Our collaborative studies have led to a multistep model for DM1 pathogenesis: (1) transcription of the mutant allele generates poly(CUG)exp RNA; (2) mutant transcripts accumulate in the nucleus in discrete foci (ribonuclear foci); (3) splicing factors in the muscleblind (MBNL) family, principally MBNL1, are sequestered in the ribonuclear foci; (4) reduced activity of MBNL1 protein leads to abnormal regulation of alternative splicing for a select group of pre-mRNAs, such as, the sarcolemmal chloride channel, ClC-1; and (5) expression of splice isoforms that are developmentally inappropriate leads to symptoms of DM1, such as, myotonia. In support of this model, we find that MBNL1 protein is recruited into ribonuclear foci so extensively that it is markedly depleted elsewhere in the nucleoplasm, and that splicing defects, or spliceopathy, in MBNL1 knockout mice are remarkably similar to those observed in DM1 patients. This model predicts that effects of DM1 in a particular nucleus are determined by levels of poly(CUG)exp accumulation in relation to supplies of MBNL1 protein, and that treatments which reduce the accumulation of poly(CUG)exp RNA or inhibit its interaction with MBNL1 have potential to stop disease progression and even accomplish a phenotypic reversal. The Aims of this proposal are to develop assays to screen for compounds that (1) reverse the spliceopathy in a cellular model of DM1; (2) inhibit the recognition of poly(CUG)exp RNA by MBNL1 protein in cells; or (3) inhibit the interaction of poly(CUG)exp RNA and MBNL1 protein in vitro. The proposed studies will be carried out in an environment that has experimental therapeutics of DM1 as its major focus. Thus, novel findings from a screening program could progress rapidly to preclinical testing in appropriate model systems and translation into therapeutic trials. Myotonic dystrophy, the most common form of muscular dystrophy in adults, causes progressive disability and premature death. Presently there are no treatments that improve the muscle weakness in people with myotonic dystrophy, or slow the disease progression. The goal of this project is to discover drugs that can be developed into effective treatments for myotonic dystrophy.