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

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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.