Using the dmd zebrafish animal model for identifying drug combination therapies and biomarkers

 描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种肌肉萎缩疾病，由DMD基因突变引起，DMD基因编码营养不良蛋白。目前还没有治愈的方法。目前的治疗标准是皮质类固醇治疗，它可以延缓骨骼肌和心脏功能障碍的进展，但也有严重的副作用。通过使用动物DMD模型，特别是MDX小鼠、GRMD狗和斑马鱼DMD突变体，已经确定了许多有前景的替代药理疗法。其中许多药物通过调节DMD基因下游的病理机制而使DMD受益。目前的挑战是确定如何最好地将临床前药理学研究转化为DMD患者。一个关键的问题是，因为许多DMD患者在服用皮质类固醇和心脏药物的同时，任何新的药物治疗都应该与现有的治疗方法进行比较，并测试其有效性。第二个问题是，治疗DMD的新型药物联合疗法的潜力尚未得到充分探索。在MDX小鼠身上进行药物比较和联合研究是可能的，但面临许多挑战。为了增加药物治疗翻译的成功概率，临床前研究应该在多个动物模型中进行验证。这项建议的长期目标是建立斑马鱼作为评估最佳DMD药物联合治疗的临床前翻译模型。这一建议的假设是斑马鱼DMD模型可用于确定改善DMD的有益药物组合和与此改善相关的生物标记物。斑马鱼为这项研究提供了几个优势，特别是可以很容易地监测心肌和骨骼肌的表型和生物标记物。这项提案将涉及两个具体目标。目的1利用斑马鱼DMD模型对一系列药物联合治疗进行系统评价。被测试的药物已经在DMD患者身上使用，或者正在进行临床试验。将检查多种结果：肌肉结构、肌肉功能、心脏结构和存活率。目的2利用qRT-PCR和RNA-seq对DMD斑马鱼进行与疾病进展和药物诱导的疾病改善相关的mRNA生物标记物的检测。该项目的一个预期影响将是表明DMD斑马鱼可以复制，因此有可能预测哺乳动物DMD药物组合效应和生物标记物。第二个潜在影响将是确定对DMD有益的新药物组合。因为这项研究将测试 对于已经在临床或临床试验中使用的药物，这些发现可能会迅速纳入患者的药物组合疗法。第三个影响是，对DMD心脏表型和心脏mRNA标记物的分析将解决DMD心脏问题的关键知识差距。该项目的长期影响将是推进一种关键的DMD动物模型，用于比较药物验证。

 DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a muscle wasting disease caused by mutations in the DMD gene, which encodes dystrophin. There are presently no cures. The current standard of care is corticosteroid treatment, which delays the progression of skeletal muscle and cardiac dysfunction but also has serious side effects. Through the use of animal DMD models, in particular the mdx mouse, the GRMD dog, and the zebrafish dmd mutant, many promising alternative pharmacological therapies have already been identified. Many of these drugs benefit DMD by modulating pathological mechanisms downstream of the DMD gene. The current challenge is to determine how to best translate preclinical pharmacological studies to DMD patients. One critical issue is that, because many DMD patients take corticosteroids as well as cardiac medication, any new pharmacological therapies should be compared to, and tested for efficacy in the presence of, these current treatments. A second issue is that the potential for novel drug combination therapies for DMD has not been fully explored. Drug comparison and combination studies are possible in the mdx mouse, but face many challenges. To increase the probability of success for translation of pharmacological therapies, preclinical studies should be validated in multiple animal models. The long-term goal of this proposal is to establish zebrafish as a preclinical translation model fo evaluating optimal DMD drug combination therapies. The hypothesis of this proposal is that the zebrafish dmd model can be used to identify both beneficial drug combinations that ameliorate DMD and biomarkers associated with this amelioration. Zebrafish offer several advantages for this study, in particular that both cardiac and skeletal muscle phenotypes and biomarkers can be easily monitored. This proposal will address two Specific Aims. Aim 1 will systematically evaluate a set of drug combination therapies using the zebrafish dmd model. The drugs to be tested are already in use on DMD patients or are in clinical trials. Multiple outcomes will be examined: muscle structure, muscle function, cardiac structure, and survival. Aim 2 will test for mRNA biomarkers associated with disease progression and drug- induced disease amelioration using qRT-PCR and RNA-seq on dmd zebrafish. One expected impact of this project will be to show that dmd zebrafish can replicate, and therefore potentially be predictive of, mammalian DMD drug combination effects and biomarkers. A second potential impact will be the identification of novel drug combinations that are beneficial for DMD. Because this study will test drugs that are already being used in the clinic or in clinical trials, the findings could potentialy be rapidly incorporated into drug-combination therapies for patients. A third impact is that the analysis of the dmd cardiac phenotype and cardiac mRNA markers will address a critical knowledge gap in DMD cardiac issues. The long-term impact of this project will be to advance a critical DMD animal model for comparative drug validation.