Therapeutic Modulation of Myotonic Muscular Dystrophy

 描述（由申请人提供）：强直性肌营养不良1型（DM 1）由一种非常规突变触发的新型RNA介导的疾病过程引起。该突变是DM蛋白激酶（DMPK）基因中CTG重复序列的不稳定扩增，其机制涉及含有扩增的CUG重复序列的转录本的毒性功能获得。研究表明，DM 1的症状发作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。为了从根本上打击DM 1，我们与业界同事合作开发了针对有毒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是否可能表现出等位基因选择性，也就是说，他们是否优先目标转录含有扩展CUG重复（CUGexp）。我们以前的工作表明，CUGexp含有转录物是超敏感的阿索敲除，由于在细胞核中的停留时间延长。我们开发了一种新的小鼠模型，用于测试ASO的等位基因歧视，基于目标转录本中是否存在扩展的CUG重复序列。将比较扩增与非扩增等位基因对阿索/RNA酶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口。这些结果将指导治疗策略的发展，以最大限度地提高和保护DM激酶的残余功能。接下来，我们将确定扩增的重复序列的体细胞不稳定性是否通过DMPK靶向ASO降低。先前我们表明CAG重复ASO能够在体内DM 1模型中稳定CTG重复。然而，由于优选的治疗策略现在已经转移到靶向重复序列之外，我们需要确定针对DMPK的非重复序列的ASO是否也可以稳定扩增的重复序列。如果ASO确实表现出减轻RNA毒性和稳定重复序列的双重性质，这将为早期开始治疗提供依据。接下来，我们将研究肌细胞中阿索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，再加上前体mRNA的定量测定，我们将确定是否长期的活性来自转录后沉默，并取决于阿索的持续存在和作用。最后，我们将确定在表达CUGexp的MBNL 1缺陷模型中，早期开始阿索治疗是否可以预防营养不良性病理学、剪接病和肌无力。总之，拟议研究的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。

 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.