Paediatrics neuromuscular disorders, genetics. Title: Effect of myonuclear domain structure on therapies for Duchenne muscular dystrophy

儿科神经肌肉疾病、遗传学。

• 批准号: 2451343
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2451343
• 关键词: Paediatrics; neuromuscular; disorders; genetics.; Title

Effect of myonuclear domain structure on therapies for Duchenne muscular dystrophy. Duchenne muscular dystrophy (DMD) is the most prevalent genetic myopathy affecting children caused by mutations which disrupt expression of the dystrophin gene. Dystrophin performs structural and signalling roles and is crucial for protecting the muscle cell membrane (sarcolemma) from contractile damage. The re-expression of dystrophin protein in muscles of DMD patients remains a significant technical challenge. Accelerated approval of exon skipping drug eteplirsen by US FDA generated controversy in the field1, since the drug was shown to restore less than 1% of normal dystrophin protein expression. Therefore, the minimal levels of dystrophin required for therapeutic benefit is a question of key importance. An allelic condition, Becker muscular dystrophy (BMD) is characterized by later onset and slower progression. In fact, some BMD patients are near-asymptomatic as a consequence of ~30% of normal dystrophin expression from birth2. Further studies from the Wood group suggest that therapeutic re-introduction of as little as ~15% of normal dystrophin in adult dystrophic (mdx) mice is sufficient to provide protection against contractile damage3. Others, suggest that homogenous expression of dystrophin at 20-30% can significantly reduce muscle pathology4. The amount, quality, and correct localization of dystrophin are fundamental issues that underlie the success of dystrophin rescue therapies. There are a plethora of approaches which aim to restore dystrophin (e.g. exon skipping, gene therapy, cell therapy, stop codon read-through, gene editing)5. Each strategy has distinct characteristics in terms of the efficacy of protein restoration, the quality (i.e. the size, or amount of truncation), and the localisation of dystrophin within the myofibre. The latter point in particular is a relatively neglected area of study. Myofibres are long syncytial structures consisting of a multitude of myoblast-derived nuclei, each serving its own proximal region of cytoplasm (i.e. myonuclear domain). To investigate differences in dystrophin localisation researchers from the Wood group have previously generated a genetic mouse model that expresses varying levels of dystrophin from birth in a mosaic pattern as a consequence of skewed X-chromosome inactivation (the mdx-Xist mouse)6. These mice exhibit a within-fibre patchy dystrophin distribution. In contrast, exon skipping therapy in mdx mice using peptide-PMO (PPMO) conjugates resulted in a uniform pattern of dystrophin expression (despite total dystrophin protein levels being similar to the mdx-Xist mice). Importantly, stabilisation of muscle turnover was observed in the PPMO-treated animals, but was still apparent in the mdx-Xist mice. These findings suggest that the pattern of dystrophin localisation is a critical factor for the correction of dystrophic pathology. Furthermore, strategies such as cell therapy and CRISPR/Cas9 are likely to also generate chimeric myofibres whereby some nuclei express dystrophin and some do not. This situation is analogous to that observed in our mdx-Xist mice, and highlights an important limitation of these therapeutic approaches. Lastly, our data strongly suggest that dystrophin mRNA and protein are not free to diffuse throughout a myofibre, but may instead be limited to the cytoplasm/sarcolemma immediately surrounding the myonuclei from which they originate, consistent with the myonuclear domain hypothesis. This project aims to characterise the heterogeneity in gene expression between dystrophin-positive and dystrophinnegative myonuclear domains contained within myofibres, and to determine the relevance of myonuclear domain structures in the context of DMD therapies. We propose to explore this area using a number of parallel approaches

肌营养结构域对Duchenne型肌营养不良症治疗的影响。 杜氏肌营养不良症（DMD）是影响儿童的最普遍的遗传性肌病，其由破坏肌营养不良蛋白基因表达的突变引起。肌营养不良蛋白执行结构和信号作用，并且对于保护肌肉细胞膜（肌膜）免受收缩损伤至关重要。肌营养不良蛋白在DMD患者肌肉中的再表达仍然是一个重大的技术挑战。美国FDA加速批准外显子跳跃药物eteplirsen在该领域引起争议1，因为该药物显示恢复不到1%的正常肌营养不良蛋白表达。因此，治疗益处所需的肌营养不良蛋白的最低水平是一个至关重要的问题。贝克肌营养不良症（BMD）是一种等位基因疾病，其特征是发病较晚，进展较慢。事实上，一些BMD患者几乎无症状，这是由于出生时约30%的正常肌营养不良蛋白表达2。Wood小组的进一步研究表明，在成年营养不良（mdx）小鼠中治疗性重新引入低至约15%的正常肌营养不良蛋白足以提供针对收缩性损伤的保护3。其他研究表明，肌营养不良蛋白的同质表达在20-30%可以显着减少肌肉病理学4。抗肌萎缩蛋白的数量、质量和正确定位是抗肌萎缩蛋白补救疗法成功的基础。有大量的方法旨在恢复抗肌萎缩蛋白（例如外显子跳跃，基因治疗，细胞治疗，终止密码子通读，基因编辑）5。每种策略在蛋白质恢复的功效、质量（即截短的大小或量）和肌纤维内肌营养不良蛋白的定位方面具有不同的特征。后一点尤其是一个相对被忽视的研究领域。肌纤维是长的合胞体结构，由大量成肌细胞衍生的细胞核组成，每个细胞核服务于其自己的细胞质近端区域（即肌纤维结构域）。为了研究肌营养不良蛋白定位的差异，Wood小组的研究人员先前已经产生了一种遗传小鼠模型，该模型从出生起就以马赛克模式表达不同水平的肌营养不良蛋白，这是由于偏斜的X染色体失活（mdx-Xist小鼠）。这些小鼠表现出纤维内斑片状肌营养不良蛋白分布。相比之下，使用肽-PMO（PPMO）缀合物对mdx小鼠进行外显子跳跃治疗，导致抗肌萎缩蛋白表达的统一模式（尽管总抗肌萎缩蛋白蛋白水平与mdx-Xist小鼠相似）。重要的是，在PPMO处理的动物中观察到肌肉转换的稳定，但在mdx-Xist小鼠中仍然明显。这些发现表明，肌营养不良蛋白定位的模式是一个关键因素，为纠正营养不良的病理。此外，细胞疗法和CRISPR/Cas9等策略也可能产生嵌合肌纤维，其中一些细胞核表达肌营养不良蛋白，而另一些则不表达。这种情况类似于在我们的mdx-Xist小鼠中观察到的情况，并突出了这些治疗方法的重要局限性。最后，我们的数据有力地表明，肌营养不良蛋白的mRNA和蛋白质是不能自由地扩散整个肌纤维，而是可能会限制在细胞质/肌膜直接周围的肌核，从他们的起源，一致的肌萎缩蛋白结构域假说。该项目旨在研究肌纤维中肌营养不良蛋白阳性和肌营养不良蛋白阴性肌萎缩蛋白结构域之间基因表达的异质性，并确定肌萎缩蛋白结构域结构在DMD治疗中的相关性。我们建议使用一些并行的方法来探索这一领域