Understanding the genesis of revertant muscle fibers using a novel muscle dystrophic reporter mouse (DmdEGFP-mdx)

Duchenne型肌营养不良症（DMD）是儿童期最常见的遗传性神经肌肉疾病，是由于DMD编码基因突变导致肌营养不良蛋白缺失而引起的。DMD的特征是进行性肌肉萎缩、呼吸和心脏功能下降以及早期死亡。在DMD患者的大多数肌肉活检标本和DMD动物模型（如mdx小鼠）中观察到一个有趣的现象。在抗肌萎缩蛋白阴性纤维的背景下，我们发现在所谓的回复纤维中有成簇的自发抗肌萎缩蛋白表达。回复纤维表达一种内部缺失、截短的抗肌萎缩蛋白，推测其对肌细胞功能具有保护作用;然而，回复突变体事件太罕见，不具有临床意义。在这里，我建议阐明导致回复突变体纤维产生的潜在细胞和分子机制，这是目前未知的。在我的博士论文中，我建立了两个转基因小鼠模型，在野生型（DmdEGFP）和mdx（DmdEGFP-mdx）遗传背景上的所谓肌营养不良蛋白-EGFP报告基因小鼠。这些模型对于研究回复纤维的发育和随后的分子分析至关重要。首先，通过分析发现，回复突变纤维可以通过天然的EGFP表达来追踪，从而验证了本文提出的研究模型。以确定回复突变体纤维中肌营养不良蛋白表达的恢复是否基于二次体细胞突变、稳定的选择性剪接或其他DNA修饰。DmdEGFP-mdx报告小鼠在外显子23中携带终止突变，因此是肌营养不良蛋白阴性的。EGFP标签仅在回复突变事件发生时表达，该回复突变事件克服了终止突变并翻译mRNA直到最后一个编码外显子79及其附加的EGFP标签。因此，我将能够分析回变肌营养不良蛋白的表达，跟踪其位置，研究这些回变纤维在体内和离体的基础上的EGFP荧光信号，并进行后续的分子分析，揭示机制和因素有助于出现和/或扩大的回变纤维可能有助于开发新的治疗DMD通过开发和增强细胞自身的机制，以恢复肌营养不良蛋白。

Duchenne muscular dystrophy (DMD), the most frequent hereditary neuromuscular disorder in childhood, is caused by the absence of dystrophin due to mutations in the encoding DMD gene. DMD is characterized by progressive muscle wasting, decline of respiratory and cardiac function and early death. A curative treatment does not exist.An intriguing phenomenon is observed in most muscle biopsy specimens of DMD patients and in DMD animal models such as the mdx mouse. On the background of dystrophin-negative fibers we find clusters of spontaneous dystrophin expression in so called revertant fibers. Revertant fibers express an internally deleted, truncated dystrophin protein, which is presumed to be protective for muscle cell function; however, the revertant event is too rare to be of clinical relevance.Here I propose to elucidate the underlying cellular and molecular mechanism leading to the generation of revertant fibers, which is currently unknown.During my PhD thesis I established two transgenic mouse models, so-called dystrophin-EGFP reporter mice on the wildtype (DmdEGFP) and mdx (DmdEGFP-mdx) genetic background. These models are pivotal for studying the development of revertant fibers and for subsequent molecular analysis. First analyses show that revertant fibers can be traced by natural EGFP expression, thereby validating the model for the herein proposed research.My main research aims are1. to determine whether the restauration of dystrophin expression in revertant fibers is based on secondary somatic mutations, on stable alternative splicing, or on other DNA modifications,2. to determine whether this revertant event takes place in muscle stem cells and whether it is inherited by muscle stem cell progenitors.DmdEGFP-mdx reporter mice carry a stop mutation in exon 23 and are hence dystrophin negative. The EGFP-tag is only expressed if a revertant event had taken place that overcomes the stop mutations and translates the mRNA until the last coding exon 79 with its appended EGFP-tag. Thus I will be able to analyze the expression of revertant dystrophin, trace its location, study these revertant fibers in vivo and ex vivo based on the EGFP fluorescent signal and perform subsequent molecular analyses.Unraveling the mechanism and factors contributing to the emergence and/or expansion of revertant fibers could contribute to the development of novel therapies for DMD by exploiting and enhancing the cells own mechanism to restore dystrophin.