Therapeutic approach targeting RNA disease in myotonic dystrophy

描述（由申请人提供）：强直性肌营养不良1型（DM 1）是最常见的肌营养不良形式，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DM 1患者的肌肉萎缩和无力，或者减缓疾病进展。这种疾病是由DMPK基因的3'非翻译区中CTG重复序列的扩增引起的。最近的研究结果表明，DM 1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。来自突变等位基因的转录物含有扩增的CUG重复序列，并且突变RNA的致病作用显然来自该poly（CUG）exp元件。例如，我们发现在完全不同的mRNA的3'非翻译区中表达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导致了DM 1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）突变转录物在细胞核中以离散灶的形式积累（3）肌盲（MBNL）家族中的剪接因子，主要是MBNL 1，被隔离在核糖核灶中;（4）MBNL 1蛋白活性的降低导致选择的一组前mRNA的选择性剪接的异常调节，例如肌膜氯离子通道ClC-1;和（5）发育不适当的剪接异构体的表达导致DM 1的症状，例如肌强直。为了支持这一模型，我们发现MBNL 1蛋白被广泛地招募到核糖核灶中，以至于在核质的其他地方明显耗尽，并且MBNL 1敲除小鼠中的剪接缺陷或剪接病与DM 1患者中观察到的剪接缺陷或剪接病非常相似。该模型预测，DM 1在特定细胞核中的作用由与MBNL 1蛋白供应相关的poly（CUG）exp积累水平决定，并且减少poly（CUG）exp RNA积累或抑制其与MBNL 1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筛选化合物的试验，这些化合物（1）逆转DM 1细胞模型中的剪接病;（2）抑制细胞中MBNL 1蛋白对poly（CUG）exp RNA的识别;或（3）抑制poly（CUG）exp RNA和MBNL 1蛋白在体外的相互作用。拟议的研究将在以DM 1实验疗法为主要重点的环境中进行。因此，筛选项目的新发现可以迅速进展到适当模型系统的临床前测试，并转化为治疗试验。强直性肌营养不良症是成人肌营养不良症中最常见的形式，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强直性肌营养不良患者的肌无力或减缓疾病进展。该项目的目标是发现可以开发成有效治疗强直性肌营养不良症的药物。

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.