Dystrophin turnover after exon-skipping

摘要 杜氏肌营养不良症（DMD）是最常见的致死性遗传性X连锁 肌病治疗这种疾病最有希望的方法是使用反义核酸。 促进破坏开放阅读框架的外显子跳跃的试剂， 转录本的可翻译形式，从而恢复缺失的肌营养不良蛋白的表达 蛋白目前对这一想法的临床试验已经给出了结果， 肌营养不良蛋白的产量和样品间变异。该提案旨在评估 将外显子跳跃与生产联系起来的过程的基本要素， 维持肌肉中抗肌萎缩蛋白的功能量。为此，我们将 研究突变型抗肌萎缩蛋白基因内外显子跳跃对 通过跳过不同的抗肌营养不良蛋白产生的3种不同的截短的抗肌营养不良蛋白的产生 外显子，并测量这些蛋白质在骨骼和 营养不良mdx小鼠的心肌。我们将使用mdx鼠标， 外显子52已经从肌营养不良蛋白基因中缺失，因为该突变呈现出一种 恢复开放阅读框和肌营养不良蛋白产生的选择数 跳过人体试验的主要目标不同的外显子。通过使用质量 我们实验室最近开发的光谱技术， 测量肌营养不良蛋白和肌营养不良蛋白相关蛋白的量， 其生产、稳定性和周转的动态。我们的目标是把这些 采取措施，以减轻与恢复3 不同的蛋白质。这将是第一次调查这类的动力学， 肌肉蛋白质，并将提供方法和协议， 更广泛适用。基于这些知识，应该可以设计出 在临床试验中将外显子跳跃应用于DMD男孩的最佳方案。它将 还提供了选择最佳外显子和外显子组合的客观方法 随着反义试剂的不断改进，

Abstract Duchenne muscular dystrophy (DMD) is the commonest lethal inherited X-linked myopathy. The most promising approach to treating this condition, is by use of antisense agents that promote skipping of exons that disrupt open reading frame so as to provide a translatable form of the transcript, thus restoring expression of the missing dystrophin protein. Current clinical trials of this idea have given results that are compromised by low yields of dystrophin and inter-sample variation. This proposal aims to evaluate the fundamental elements of the processes that link exon-skipping to the production and maintenance of functional amounts of dystrophin in muscles. To do this we will investigate the effects of exon-skipping within the mutant dystrophin gene on the production of the 3 different truncated dystrophin proteins produced by skipping different exons and measure the stability and longevity of these proteins within skeletal and cardiac muscles of the dystrophic mdx mouse. We will use the mdx mouse in which exon 52 has been deleted from the dystrophin gene because this mutation presents a number of options for restoring open reading frame and dystrophin production by skipping of different exons that are the main targets of human trials. By use of Mass Spectroscopy techniques recently developed within our laboratory we can accurately measuring the amounts of dystrophin and dystrophin-associated proteins, and will follow the dynamics of their production, stability and turnover. Our aim is to relate these measures to the extent of alleviation of pathology associated with the restoration of 3 different these proteins. This will be the first investigation of the dynamics of this class of muscle proteins in muscles in vivo and will provide methods and protocols that will be more widely applicable. On the basis of this knowledge it should be possible to devise optimal protocols for application of exon-skipping to DMD boys in clinical trials. It will also provide an objective method of selecting the best exons and combinations of exons to skip as the progressively improving antisense reagents become available.