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3,500个男孩中就有1个受到影响。来自多个实验室的累积证据证实了钙失调和氧化应激是该疾病的关键贡献者，这表明钙螯合（CaSeq）或抗氧化剂（Antiox）途径的上调可以作为DMD治疗的靶点。本申请旨在鉴定对营养不良表型最重要的CaSeq/Antiox途径，并评估可以通过设计用于靶向这些途径的基因疗法实现的治疗潜力。该项目有三个具体目标，并将利用两种DMD小鼠模型：mdx和mdx：utrn-/-菌株。在目的1中，将使用肌细胞和分离的肌肉制备物表征单个CaSeq/Antiox途径对营养不良表型的影响。具有最大影响的CaSeq/Antiox途径将被用作病毒介导的基因疗法的靶点。为了评估这些基因疗法的功效，营养不良小鼠将静脉内注射含有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，并纠正与肌营养不良蛋白缺乏相关的病理生理学。虽然本申请的范围仍然集中在DMD上，但我们希望我们的研究结果的治疗方面与钙失调或氧化应激起关键作用的其他疾病的治疗直接相关，例如阿尔茨海默病，衰老，糖尿病和心血管疾病。在本申请的最终目的中，我们通过将mdx小鼠与具有修饰的CaSeq/Antiox途径的现有小鼠品系杂交来产生具有修饰的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.