Therapeutic Modulation of Myotonic Muscular Dystrophy

强直性肌营养不良症的治疗调节

• 批准号: 9984584
• 负责人: CHARLES A THORNTON
• 金额: $ 37.06万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9984584
• 关键词: 3' Untranslated Regions; Ablation; Affect; Aftercare; Age; Alleles; Alternative Splicing; Antisense Oligonucleotides; Binding Proteins; Biochemical; Biogenesis; Biological; Biological Assay; CAG repeat; Cardiac; Cell Culture Techniques; Cell Extracts; Cell Nucleus; Clinical Trials; Coupled; Cytoplasm; DNA; DNA Repair; Defect; Development; Discrimination; Disease; Dose; Exhibits; Exonuclease; Gene Frequency; Gene Silencing; Genes; Genetic Transcription; Goals; Growth; Human; Hypersensitivity; Individual; Industry; Injections; Investigational Therapies; Laboratories; Learning; Macaca fascicularis; Mediating; Modeling; Mus; Muscle; Muscle Cells; Muscle Fibers; Muscle Weakness; Muscular Dystrophies; Mutation; Myocardium; Myopathy; Myotonia; Myotonic Dystrophy; Myotonic Muscular Dystrophy; Nuclear; Oligonucleotides; Output; Pathology; Pharmaceutical Preparations; Plant Roots; Process; Property; Proteins; RNA; RNA Splicing; Reagent; Refractory; Regimen; Residual state; Ribonuclease H; Safety; Single-Stranded DNA; Site; Skeletal Muscle; Somatic Cell; Subcutaneous Injections; Symptoms; Testing; Therapeutic; Therapeutic Trials; Time; Tissue Sample; Tissues; Toxic effect; Transcript; Transgenic Mice; Work; age related; base; clinical practice; design; gain of function; in vivo Model; knock-down; mRNA Precursor; mouse model; mutant; myotonic dystrophy protein kinase; novel; postnatal; preservation; prevent; programs; public health relevance; systemic toxicity; therapeutic candidate; therapeutic development

 DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1) results from a novel RNA-mediated disease process that is triggered by an unconventional mutation. The mutation is an unstable expansion of CTG repeats in the DM protein kinase (DMPK) gene, and the mechanism involves a toxic gain-of-function by transcripts containing an expanded CUG repeat. Studies indicate that symptom onset and progression of DM1 is driven by the age-dependent growth of the expanded CTG repeat in somatic cells. In an effort to attack DM1 at its root cause, we have partnered with colleagues in industry to develop antisense oligonucleotides (ASOs) targeting the toxic RNA. This approach was highly effective in mouse models and has recently entered clinical trials. In this proposal we seek to better define the therapeutic properties of these versatile new reagents. First we will determine whether ASOs may exhibit allelic selectively; that is, whether they preferentially target transcripts containing expanded CUG repeats (CUGexp). Our previous work suggested that CUGexp-containing transcripts are hypersensitive to ASO knockdown, owing to prolonged dwell time in the nucleus. We developed a new mouse model for testing the allelic discrimination of ASOs, based on the presence or absence of expanded CUG repeats in the target transcript. The sensitivity of expanded vs. non-expanded alleles to ASO/RNase H knockdown will be compared, and the therapeutic window for allelic selectivity will be determined for different dosing regimens. The results will guide th development of therapeutic strategies to maximize and preserve the residual function of the DM kinase. Next we will determine whether somatic instability of expanded repeats is reduced by DMPK-targeting ASOs. Previously we showed that CAG-repeat ASOs are able to stabilize CTG repeats in a DM1 model in vivo. However, as the preferred therapeutic strategy has now shifted to targeting outside of the repeat tract, we need to determine whether ASOs directed at the non-repetitive sequences of DMPK may also stabilize expanded repeats. If ASOs do in fact exhibit dual properties of mitigating RNA toxicity and stabilizing repeats, this will argue for early initiation of treatment. Next, we will examine the biochemical basis for the unusually prolonged duration of ASO activity in muscle cells. Using decoy oligonucleotides that are complementary to ASOs, coupled with quantitative assays of pre-mRNA, we will determine whether long-duration activity results from post-transcriptional silencing and depends on the continuous presence and action of the ASO. Finally, we will determine whether early initiation of ASO treatment can prevent dystrophic pathology, spliceopathy, and muscle weakness in a CUGexp-expressing, MBNL1-deficient model. Taken together, the results of the proposed studies will expand our understanding of the biological properties of ASOs in skeletal muscle, and provide important guidance for the optimal use of these reagents in therapeutic trials and clinical practice.

 描述（由申请人提供）：1 型强直性肌营养不良 (DM1) 是由一种非常规突变触发的新型 RNA 介导的疾病过程引起的。该突变是 DM 蛋白激酶 (DMPK) 基因中 CTG 重复的不稳定扩展，其机制涉及包含扩展的 CUG 重复的转录本的毒性功能获得。研究表明，DM1 症状的发生和进展是由体细胞中扩增的 CTG 重复序列的年龄依赖性生长驱动的。为了从根源上攻击 DM1，我们与业界同行合作开发针对有毒 RNA 的反义寡核苷酸 (ASO)。这种方法在小鼠模型中非常有效，最近已进入临床试验。在本提案中，我们寻求更好地定义这些多功能新试剂的治疗特性。首先，我们将确定 ASO 是否可以选择性地表现出等位基因；也就是说，它们是否优先靶向包含扩展 CUG 重复序列 (CUGexp) 的转录本。我们之前的工作表明，由于在细胞核中的停留时间较长，含有 CUGexp 的转录本对 ASO 敲低非常敏感。我们开发了一种新的小鼠模型，用于根据目标转录本中是否存在扩展的 CUG 重复来测试 ASO 的等位基因区分。将比较扩增与非扩增等位基因对 ASO/RNase H 敲低的敏感性，并将确定不同给药方案的等位基因选择性的治疗窗。结果将指导治疗策略的开发，以最大化和保留 DM 激酶的残余功能。接下来我们将确定 DMPK 靶向 ASO 是否可以降低扩展重复的体细胞不稳定性。之前我们证明了 CAG 重复 ASO 能够在体内 DM1 模型中稳定 CTG 重复。然而，由于首选的治疗策略现在已转向重复序列之外的靶向，我们需要确定针对 DMPK 非重复序列的 ASO 是否也可以稳定扩展的重复序列。如果 ASO 实际上表现出减轻 RNA 毒性和稳定重复序列的双重特性，那么这将为尽早开始治疗提供理由。接下来，我们将研究肌肉细胞中 ASO 活性持续时间异常延长的生化基础。使用与 ASO 互补的诱饵寡核苷酸，结合前 mRNA 的定量分析，我们将确定长期活性是否由转录后沉默导致，并取决于 ASO 的持续存在和作用。最后，我们将确定早期开始 ASO 治疗是否可以预防表达 CUGexp 的 MBNL1 缺陷模型中的营养不良病理学、剪接病和肌肉无力。总而言之，所提出的研究结果将扩大我们对骨骼肌中 ASO 生物学特性的理解，并为这些试剂在治疗试验和临床实践中的最佳使用提供重要指导。