PKR as a therapeutic target for muscular dystrophy

项目概要/摘要。杜氏肌营养不良症（DMD）是最常见的，致命的，X连锁疾病 国际吧进行性肌无力导致生命早期的骨骼肌丧失，随后导致死亡， 呼吸或心脏衰竭。营养不良的肌肉在如此年轻的时候对严重损伤的易感性， 最近的临床试验的缺点强调了迫切需要确定有效的策略来保护mus， DMD患者的循环功能。抗炎药物提供有限的，但有意义的，治疗效果， 但伴随着副作用。另外，内质网（ER）应激正在成为一个调解人 疾病的发展。双链RNA依赖性蛋白激酶（PKR）是肌肉的有力驱动力， 炎症和ER应激，并且在人类和啮齿动物营养不良骨骼肌中升高。PKR抑制 在几种肌肉病变中发挥强大的抗炎作用并防止ER应激。因此，委员会认为， 研究PKR在疾病进展中的作用以及作为未来治疗靶点的重要性 关于DMD我们的长期目标是制定和实施成功预防或治疗疾病的战略- DMD的相关病理。本申请的目的是确定PKR作为以下启动子的作用： 肌营养不良蛋白缺乏骨骼肌中炎症、ER应激和肌肉功能障碍。我们的核心假设 PKR抑制将减弱肌营养不良蛋白缺陷型中的炎症、ER应激和疾病参数， 骨骼肌。这一假设是根据现有文献和我们自己的初步发现制定的 证明：1）DMD患者肌肉中PKR表达增加， mdx小鼠肌肉; 2）肌营养不良蛋白缺乏伴随肌肉炎症和ER应激; 3）PKR 抑制减弱肌肉炎症、ER应激和萎缩，包括糖皮质激素;和4）PKR β 1， 它可以防止炎症引起的肌肉功能障碍。在Aim 1中，PKR将被使用腺- 相关病毒（AAV）介导的靶向PKR mRNA的siRNA递送。我们将在体内和体外测量 功能参数，然后在mdx和对照小鼠中进行生化和组织病理学分析， 没有糖皮质激素治疗。我们假设PKR的敲低会减弱炎症和ER， 压力以及肌肉损伤和功能障碍。在目标2中，PKR抑制剂伊莫辛将与或 不使用糖皮质激素，对营养不良的小鼠进行4个月，随后测量体内肢体肌肉力量 和呼吸功能、体外肌肉功能和组织病理学。我们假设伊莫辛会减弱 肌营养不良蛋白缺乏的骨骼肌的肌肉损伤和功能障碍，伴随着炎症反应的减少， 和ER应力。这个项目是创新的，因为它采用了一种新颖的方法，以前没有考虑过， 对抗由肌营养不良蛋白缺乏引起的肌肉损伤。这一贡献意义重大，因为它将 它将解决一个关键的知识差距，有关机制的病理生理学引起的 肌营养不良蛋白缺乏症，并提出了用于治疗肌营养不良症的新的治疗靶点。

Project Summary/Abstract. Duchenne muscular dystrophy (DMD) is the most common, fatal, X-linked disease worldwide. Progressive muscle weakness leads to loss of ambulation early in life, followed by death caused by respiratory or cardiac failure. The susceptibility of dystrophic muscle to severe injury at such a young age, and shortcomings of recent clinical trials underscore the urgent need to identify effective strategies to preserve mus- cle function in DMD patients. Anti-inflammatory drugs provide limited, though meaningful, therapeutic impact, but are accompanied by side effects. Separately, endoplasmic reticulum (ER) stress is emerging as a mediator of disease progression. The double-stranded RNA-dependent protein kinase (PKR) is a potent driver of muscle inflammation and ER stress, and is elevated in human and rodent dystrophic skeletal muscle. Inhibition of PKR elicits powerful anti-inflammatory effects and prevents ER stress in several muscle pathologies. Consequently, there is a critical need to investigate the role of PKR in disease progression and as a future therapeutic target for DMD. Our long-term goal is to develop and implement strategies to successfully prevent or treat disease- related pathologies in DMD. The objective in this application is to determine the role of PKR as a promotor of inflammation, ER stress, and muscle dysfunction in dystrophin-deficient skeletal muscle. Our central hypothesis is that inhibition of PKR will attenuate inflammation, ER stress, and disease parameters in dystrophin-deficient skeletal muscle. This hypothesis was formulated based on existing literature and our own preliminary findings demonstrating that: 1) PKR expression is increased in muscle of DMD patients and activation is elevated in muscle from mdx mice; 2) dystrophin deficiency is accompanied by muscle inflammation and ER stress; 3) PKR inhibition attenuates muscle inflammation, ER stress, and atrophy, including glucocorticoids; and 4) PKR inhibi- tion protects against inflammation-induced muscle dysfunction. In Aim 1 PKR will be knocked down using adeno- associated virus (AAV)-mediated delivery of siRNA targeting PKR mRNA. We will measure in vivo and in vitro functional parameters followed by a biochemical and histopathological analysis in mdx and control mice with our without glucocorticoid treatment. We hypothesize that knockdown of PKR will attenuate inflammation and ER stress as well as muscle injury and dysfunction. In Aim 2 the PKR inhibitor imoxin will be administered, with or without glucocorticoid, to dystrophic mice for four months followed by measures of in vivo limb muscle strength and respiratory function, in vitro muscle function, and histopathology. We hypothesize that imoxin will attenuate muscle injury and dysfunction in dystrophin-deficient skeletal muscle, concomitant with reductions in inflamma- tion and ER stress. This project is innovative because it utilizes a novel approach, not previously considered, to combat the muscle impairments caused by dystrophin deficiency. This contribution is significant because it will it will address a critical knowledge gap regarding the mechanisms underlying the pathophysiology caused by dystrophin deficiency and suggest a novel therapeutic target for the treatment of muscular dystrophy.