RNA-mediated mechanisms in the myotonic dystrophies

强直性肌营养不良中 RNA 介导的机制

• 批准号: 7917325
• 负责人: CHARLES A THORNTON
• 金额: $ 32.12万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917325
• 关键词: 3' Untranslated Regions; Adult; Affect; Alleles; Alternative Splicing; Antisense Oligonucleotides; Binding; Binding Proteins; Biochemical; Biological Markers; CAG repeat; Candidate Disease Gene; Cell Nucleus; Cell physiology; Cells; Cessation of life; Chemistry; Clinical; Clinical Research; Clinical Trials; Collaborations; Complex; Custom; Data; Defect; Disease; Distal; Exons; Family; Fingers; Flexor; Foundations; Genes; Genetic Predisposition to Disease; Genetic Transcription; Hereditary Disease; Human Genetics; In Vitro; Individual; Introns; Investigation; Knowledge; Lead; Length; Longitudinal Studies; Mediating; Messenger RNA; Methods; Modeling; Muscle; Muscular Dystrophies; Mutation; Myotonia; Myotonic Dystrophy; Nuclear; Oligonucleotides; Pathogenesis; Patients; Peptide Nucleic Acids; Phenotype; Physiological; Process; Prospective Studies; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA amplification; Regulation; Research; Sampling; Severity of illness; Site; Symptoms; Testing; Therapeutic; Therapeutic Agents; Transgenic Mice; Translating; Untranslated Regions; Work; cohort; cost; design; disability; gain of function; insight; mouse model; mutant; novel; premature; prospective; response; tibialis anterior muscle; tool; wasting

DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1), the most prevalent form of muscular dystrophy in adults, leads to progressive disability and premature death. No treatment that alters the course of DM1 is presently available. This disorder is caused by expansion of a CTG repeat in the 34 untranslated region of the DMPK gene. This unusual mutation leads to a novel, RNA-mediated disease process. Our working model for DM1 pathogenesis involves the following steps: (1) transcription of the mutant allele generates RNA with an expanded CUG repeat (CUGexp); (2) CUGexp RNA accumulates in the nucleus in discrete foci; (3) splicing factors in the muscleblind (MBNL) family, principally MBNL1, are sequestered in the nuclear foci; (4) loss of MBNL1 function leads to abnormal regulation of alternative splicing for a select group of pre-mRNAs; and (5) expression of incorrect splice isoforms leads to symptoms of DM1. A second type of myotonic dystrophy, DM2, has been identified, and it shares a similar RNA-mediated pathogenesis. Misregulated alternative splicing, or spliceopathy, is the major biochemical derangement that is currently recognized in DM1 or DM2. It is clear that spliceopathy can explain certain aspects of the phenotype, such as, myotonia, but the range of genes that are affected, and how the spliceopathy relates to the full spectrum of clinical features, is not clearly defined. To pursue this question, our first Aim is to use splicing sensitive microarrays to analyze spliceopathy in DM1 and DM2. We will obtain a comprehensive view of spliceopathy, and then apply this knowledge to create a custom, cost-effective array that is specifically designed to assess DM-related splicing changes. This tool will then be applied in a prospective study of DM1 and DM2 patients. This study will identify candidates for involvement in the muscle wasting of DM1. Furthermore, the validity of spliceopathy as a biomarker for disease severity and therapeutic response will be tested. Our second aim is to translate recent mechanistic insights into new treatments for patients with DM1. We propose a novel use of antisense oligonucleotides, namely, to bind to CUGexp RNA and displace sequestered proteins, thereby releasing these proteins to carry out their normal functions. Preliminary data indicate that a CAG-repeat oligonucleotide has this intended effect, and that physiological and biochemical signs of DM1 can be reversed by this strategy in a transgenic mouse model. To develop the therapeutic potential of CAG-repeat oligos, we will test oligonucleotides of different lengths and chemistries for their ability to interact with CUGexp RNA, to disrupt complexes of MBNL1-CUGexp in vitro, to inhibit the interaction of MBNL1 protein with CUGexp RNA in cells, and to reverse the spliceopathy and myotonia in a transgenic mouse model of DM. Methods for systemic delivery of these oligonucleotides will be tested, and potential off-target effects will be examined. This work may lead to the first treatment that is capable of reversing the symptoms of DM1.

描述（由申请人提供）：成年人中最普遍的肌肉营养不良形式1型肌发育症（DM1）导致渐进的残疾和早亡。目前可以使用改变DM1过程的治疗方法。这种疾病是由于DMPK基因的34个未翻译区域中CTG重复的扩展引起的。这种不寻常的突变导致了一种新型的RNA介导的疾病过程。我们用于DM1发病机理的工作模型涉及以下步骤：（1）突变等位基因的转录产生RNA，并具有扩展的CUG重复（CugeXP）； （2）CugeXP RNA在离散灶中积聚在核中； （3）肌肉闪光（MBNL）家族中的剪接因子（主要是MBNL1）在核灶中被隔离； （4）MBNL1函数的丧失导致选择一组MRNA的替代剪接的异常调节； （5）不正确的剪接同工型的表达导致DM1的症状。已鉴定出第二种类型的肌发育症DM2，并具有类似的RNA介导的发病机理。替代剪接或剪接病的错误调节是目前在DM1或DM2中识别的主要生化危险。显然，剪接病可以解释表型的某些方面，例如，肌瘤，但是受影响的基因范围，以及剪接症与临床特征的全部范围之间的关系。要解决这个问题，我们的第一个目的是使用剪接敏感的微阵列来分析DM1和DM2中的剪接病。我们将获得剪接疗法的全面视图，然后应用此知识来创建一个自定义，具有成本效益的数组，该数组是专门设计用于评估与DM相关的剪接更改的。然后，该工具将应用于对DM1和DM2患者的前瞻性研究。这项研究将确定候选人参与DM1的肌肉浪费。此外，将测试剪接病作为疾病严重程度和治疗反应的生物标志物的有效性。我们的第二个目的是将最近的机械见解转化为DM1患者的新疗法。我们提出了一种新型的反义寡核苷酸的使用，即与CugeXP RNA结合并置换了隔离的蛋白质，从而释放了这些蛋白质以执行其正常功能。初步数据表明，CAG重复寡核苷酸具有这种预期效果，并且DM1的生理和生化迹象可以在转基因小鼠模型中被该策略反转。 To develop the therapeutic potential of CAG-repeat oligos, we will test oligonucleotides of different lengths and chemistries for their ability to interact with CUGexp RNA, to disrupt complexes of MBNL1-CUGexp in vitro, to inhibit the interaction of MBNL1 protein with CUGexp RNA in cells, and to reverse the spliceopathy and myotonia in a transgenic mouse model of DM。将测试这些寡核苷酸的全身传递方法，并将检查潜在的脱靶效应。这项工作可能导致第一种能够逆转DM1症状的治疗方法。