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

杜氏肌营养不良症（DMD）是儿童期最常见的遗传性神经肌肉疾病，是由于编码DMD基因的突变导致肌营养不良蛋白缺失而引起的。DMD的特点是进行性肌肉萎缩，呼吸和心脏功能下降和早期死亡。治愈的治疗方法并不存在。在大多数DMD患者的肌肉活检标本和DMD动物模型（如mdx小鼠）中观察到一个有趣的现象。在肌营养不良蛋白阴性纤维的背景下，我们在所谓的反向纤维中发现了自发的肌营养不良蛋白表达簇。可逆纤维表达一种内部缺失的、截断的肌营养不良蛋白，该蛋白被认为对肌肉细胞功能具有保护作用；然而，逆转事件太罕见，不具有临床相关性。在这里，我建议阐明导致反向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，我在野生型（DmdEGFP）和mdx （DmdEGFP-mdx）遗传背景下建立了两种转基因小鼠模型，即所谓的抗肌萎缩蛋白- egfp报告小鼠。这些模型对于研究反射纤维的发展和随后的分子分析是至关重要的。首先分析表明，反向纤维可以通过天然EGFP表达来追踪，从而验证了本文提出的研究模型。我的主要研究目标是1。以确定反向纤维中肌营养不良蛋白表达的恢复是基于次生体细胞突变、稳定的选择性剪接还是其他DNA修饰。确定这种逆转事件是否发生在肌肉干细胞中，以及是否由肌肉干细胞祖细胞遗传。DmdEGFP-mdx报告小鼠在23号外显子携带一个停止突变，因此是肌营养不良蛋白阴性。egfp -标签仅在发生逆转事件时表达，该事件克服了停止突变并翻译mRNA直到最后一个编码外显子79及其附加的egfp -标签。这样我就可以分析可逆肌营养不良蛋白的表达，追踪其位置，根据EGFP荧光信号在体内和离体研究这些可逆纤维，并进行后续的分子分析。揭示导致逆转纤维出现和/或扩张的机制和因素，可以通过开发和增强细胞自身恢复肌营养不良蛋白的机制，促进DMD新疗法的发展。

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.