Pathogenesis and Treatment of Muscular Dystrophy

描述(申请人提供)：在过去的5年里，我们一直致力于建立斑马鱼作为肌肉营养不良症的模型。在这个能力，我们已经发表了斑马鱼缺乏营养不良蛋白和5-肌聚糖的表型，完成了一个大的遗传筛选，以分离更多的营养不良突变，并在Runzel突变中鉴定了突变基因。利用我们在肌肉研究和建立斑马鱼作为疾病模型方面的经验，我们现在建议使用斑马鱼来研究肌肉营养不良的发病机制，并评估细胞疗法作为一种潜在的治疗方案。 该项目的第一个目标是全面描述我们现有的营养不良斑马鱼模型之一，目的是更好地理解肌肉营养不良的发病机制。我们选择了emz突变体进行进一步分析，因为它的突变粗略地映射到一个基因组区间，该区间没有任何与目前与肌肉营养不良相关的基因同源。这个突变体的表型与dystrophin突变体非常相似，这表明肌肉变性的emz表型是肌营养不良的症状。我们建议使用传统的作图方法来识别该突变体中的基因突变，然后对候选基因进行测序来识别特定的突变。如果人类的同源基因目前与肌营养不良症没有关联，该基因将被认为是一种候选疾病，并在肌肉营养不良症的原因未知的人类患者中进行测序。由于看似无关的蛋白质的突变可以表现为肌肉营养不良，识别额外的基因将有助于建立疾病途径。 其次，我们已经建立了在斑马鱼所有发育阶段移植细胞群体的方法，现在建议使用这个系统来识别最有能力植入和矫正患病肌肉的细胞群体。肌肉移植细胞群体的基因表达谱将与非移植细胞进行比较，以确定主要在移植细胞中表达的基因。差异表达的基因将被认为是潜在的标记，并用于纯化哺乳动物中类似的细胞群，用于未来的实验和治疗。最后，我们计划通过分析斑马鱼肌肉前体细胞的发育潜力来剖析不同干细胞群体的谱系关系。这将通过在发育早期移植有限数量的标记细胞，然后随着鱼的成熟而跟踪它们的命运来实现。

Description (provided by applicant): Over the last 5 years, we have been working to establish the zebrafish as a model for muscular dystrophy. In this capacity, we have published the phenotype of zebrafish lacking dystrophin and 5- sarcoglycan, completed a large genetic screen to isolate additional dystrophic mutants, and identified the mutant gene in the runzel mutant. Using our experiences in muscle research and in establishing the zebrafish as a disease model, we now propose to use the fish to investigate the pathogenesis of muscular dystrophy and evaluate cell therapy as a potential treatment option. The first aim in this project proposes to fully characterize one of our available dystrophic zebrafish models with the goal of better understanding the pathogenesis of muscular dystrophy. We have selected the emz mutant for further analysis since its mutation rough maps to a genomic interval void of any genes orthologous to those currently associated with muscular dystrophy. This mutant shows a phenotype very similar to the dystrophin mutant (sapje) suggesting that the emz phenotype of muscle degeneration is symptomatic of muscular dystrophy. We propose to identify the genetic mutation in this mutant using a traditional mapping approach and then sequencing candidate genes to identify the specific mutation. If the orthologous human gene is not currently associated with muscular dystrophy, the gene will be considered a disease candidate and sequenced in human patients for which the cause of muscular dystrophy is unknown. Since mutations in seemingly unrelated proteins can manifest as muscular dystrophy, the identification of additional genes would be helpful for establishing disease pathways. Secondly, we have established, methods to transplant cell populations in zebrafish at all developmental stages and now propose using this system to identify the cell population most capable of engrafting into and correcting the diseased muscle. Gene expression profiles of muscle engrafting cell populations will be compared with non-engrafting cells to identify genes expressed predominantly in the engrafting cells. Differentially expressed genes will be considered potential markers and used to purify analogous cell populations in mammals for future experimentation and therapy. Finally, we plan to dissect the lineage relationship of various stem cell populations by assaying the developmental potential of zebrafish muscle progenitor cells. This will be accomplished by transplanting limited populations of labeled cells early in development and then following their fate as the fish matures.