Therapeutic Modulation of Myotonic Muscular Dystrophy

 描述(由申请人提供)：强直性肌营养不良1型(DM1)是由非传统突变引发的一种新的RNA介导的疾病过程所致。这种突变是DM蛋白激酶(DMPK)基因中CTG重复序列的不稳定扩张，其机制涉及含有扩展的CUG重复序列的转录本的毒性功能获得。研究表明，DM1症状的发生和发展是由体细胞中扩大的CTG重复序列的年龄相关生长驱动的。为了从根本上治疗DM1，我们与工业界的同事合作，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。在这项提案中，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是否可能选择性地表现出等位基因；也就是说，它们是否优先以含有扩展的CUG重复序列(CUGexp)的转录本为靶标。我们以前的工作表明，含有CUGexp的转录本对ASO基因敲除高度敏感，这是由于其在核中的停留时间延长。我们开发了一种新的小鼠模型来测试ASOS的等位基因识别能力，该模型基于目标转录本中是否存在扩展的CUG重复序列。将比较扩展等位基因和非扩展等位基因对ASO/RNaseH基因敲除的敏感性，并将确定不同给药方案的等位基因选择性治疗窗口。这一结果将指导治疗策略的发展，以最大限度地保护DM激酶的残留功能。下一步，我们将确定DMPK靶向ASO是否降低了扩展重复序列的躯体不稳定性。在此之前，我们已经证明CAG-Repeat ASO能够在体内DM1模型中稳定CTG重复序列。然而，随着首选的治疗策略现在已经转移到重复区外靶向，我们需要确定针对DMPK的非重复序列的ASO是否也可以稳定扩大的重复序列。如果ASO确实表现出减轻RNA毒性和稳定重复序列的双重特性，这将证明应该尽早开始治疗。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互补的诱骗寡核苷酸，再加上对前mRNA的定量分析，我们将确定长时间的活动是否是转录后沉默的结果，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CUGexp表达的MBNL1缺陷模型中的营养不良病理、剪接病变和肌肉无力。综上所述，这些研究结果将扩大我们对骨骼肌中ASOS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优化使用提供重要的指导。

 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.