Calcium and oxidative stress in muscular dystrophy

杜氏肌营养不良症（DMD）是一种致命的遗传性疾病，每3500名男孩中就有1人患病。来自多个实验室的越来越多的证据证实了钙失调和氧化应激是该疾病的关键因素，这表明钙隔离（CaSeq）或抗氧化（Antiox）途径的上调可能是治疗DMD的靶点。本申请旨在确定对营养不良表型最重要的CaSeq/Antiox途径，并评估通过针对这些途径设计的基因疗法可以实现的治疗潜力。该项目有三个具体目标，并将利用两种小鼠DMD模型：mdx和mdx:utrn-/-菌株。在Aim 1中，肌肉细胞和分离的肌肉制剂将被用来表征单个CaSeq/Antiox通路对营养不良表型的影响。影响最大的CaSeq/Antiox通路将被用作病毒介导的基因治疗的靶标。为了评估这些基因疗法的疗效，将向营养不良小鼠静脉注射含有CaSeq/Antiox转基因的重组腺相关病毒。我们将确定这些转基因是否可以延长营养不良小鼠的寿命并纠正与营养不良蛋白缺乏症相关的病理生理。虽然应用范围仍然集中在DMD上，但我们希望我们的研究结果在治疗方面与其他疾病有直接的相关性，其中钙失调或氧化应激起关键作用，如阿尔茨海默病、衰老、糖尿病和心血管疾病。在应用程序的最终目的中，我们通过将mdx小鼠与具有修饰的CaSeq/Antiox途径的现有小鼠株杂交，产生具有修饰的CaSeq/Antiox途径的突变mdx小鼠。这些突变mdx小鼠将是对当前营养不良小鼠模型的有价值的补充，因为它们允许研究人员分离特定CaSeq/Antiox途径对营养不良表型的贡献。

Duchenne muscular dystrophy (DMD) is a lethal genetic disease that affects 1 in 3,500 boys. Accumulating evidence from multiple laboratories corroborate on the involvement of calcium misregulation and oxidative stress as key contributors to the disease, suggesting that upregulation of calcium-sequestering (CaSeq) or anti-oxidant (Antiox) pathways may serve as targets in the treament of DMD. The present application aims to identify the CaSeq/Antiox pathways most significant to the dystrophic phenotype, and assess the therapeutic potential that can be realized by a gene therapy designed to target these pathways. The project is framed by three specific aims and will utilize two murine models of DMD: the mdx and mdx:utrn-/- strains. In Aim 1, muscle cells and isolated muscle preparations will be used characterize the impact of individual CaSeq/Antiox pathways on the dystrophic phenotype. CaSeq/Antiox pathways with the most substantial impact will then be used as targets for viral-mediated gene therapies. To evaluate the efficacy of these gene therapies, dystrophic mice will be injected intravenously with recombinant adeno-associated viruses that contain CaSeq/Antiox transgenes. We will determine whether these transgenes can extend the lifespan of dystrophic mice and correct the pathophyosiology associated with dystrophin-deficiency. Although the scope of the application remains focused on DMD, we expect the therapeutic aspect of our findings to have a direct relevance in the treatment of other diseases where calcium misregulation or oxidative stress play a key role, such as Alzheimer's Disease, aging, diabetes and cardiovascular disease. In the final Aim of the application, we generate mutant mdx mice with modified CaSeq/Antiox pathways by crossing mdx mice with existing strains of mice that possess modified CaSeq/Antiox pathways. These mutant mdx mice will be valuable additions to current dystrophic mouse models, as they allow investigators to isolate the contribution of specific CaSeq/Antiox pathways to the dystrophic phenotype.