Mechanisms of mineralocorticoid receptor antagonism on inflammation in muscular dystrophy

项目摘要 杜氏肌营养不良症（DMD）的基因治疗目前正在年轻人的临床试验中进行测试。 患者然而，腺相关病毒（AAV）递送系统的局限性，包括其小的携带量， 能力和其低效率的肌肉干细胞转染，将仍然是一个障碍，以治愈。的 基于贝克尔肌营养不良症肌营养不良蛋白（微- 肌营养不良蛋白）仍然会导致一些骨骼肌更新，随后出现炎症和心肌病。 此外，正常肌肉使用造成的损伤将用肌肉干细胞修复， 表达抗肌萎缩蛋白。这两个问题至少会导致低水平的慢性炎症， 加剧肌肉损伤和最终的转基因表达损失，限制了功效。强的松， 作为DMD的标准治疗，但有许多严重的副作用，继续作为抗 炎性的，以防止对转基因的免疫应答。公布的数据和初步数据支持 科学前提是，对DMD有临床益处的盐皮质激素受体（MR）拮抗剂 心肌病，稳定肌膜，提高骨骼肌力量，减少纤维化，也是 是一种理想的与基因转移结合的药物。然而，反- 尚未探索肌营养不良症中MR拮抗剂的炎性特性。自从强的松 直接与MR拮抗剂竞争结合其受体，这些研究对于MR的临床应用至关重要 拮抗剂作为替代泼尼松的辅助治疗。在这个应用中，我们将测试强的松是否 和MR拮抗剂对营养不良性疾病中的特异性免疫细胞群具有相同或不同的作用。 肌肉，MR拮抗剂是否减少细胞因子依赖于MR信号传导机制， 肌纤维或骨髓细胞，以及MR拮抗剂是否限制急性损伤后的累积损伤， 用微小肌营养不良蛋白基因疗法治疗的营养不良小鼠。我们已经开发出了免疫流排序的方法 来自DMD的mdx基因型模型的单个肌肉的细胞群，其将允许首次鉴定 泼尼松抑制的免疫细胞群，尽管几十年的临床使用， 与MR拮抗剂比较。这些方法也将允许基因的无偏鉴定 MR拮抗剂和泼尼松诱导的炎性髓样细胞表达变化。这些研究 将为MR拮抗剂作为联合治疗的最佳临床用途提供信息，以延长新兴遗传疗法的疗效 治疗DMD和其他可能的肌肉萎缩症。生成的数据还将识别新的 潜在的抗炎治疗靶点。

PROJECT SUMMARY Gene therapy for Duchenne Muscular Dystrophy (DMD) is currently being tested in clinical trials in young patients. However, limitations of the adeno-associated virus (AAV) delivery system, including its small carrying capacity and its low efficiency transfection of muscle stem cells, will remain a barrier to a cure. The miniaturized transgene being delivered that is based on a Becker muscular dystrophy dystrophin (micro- dystrophin) will still result in some skeletal muscle turnover with subsequent inflammation and cardiomyopathy. Additionally, injury resulting from normal muscle use will be repaired with muscle stem cells that will likely not express dystrophin. Both of these issues will result in at least low-level chronic inflammation, which will likely exacerbate muscle damage and ultimate loss of transgene expression, limiting efficacy. Prednisone, which has served as the standard of care for DMD, but has many severe side effects, continues to be given as an anti- inflammatory to prevent an immune response to the transgene. Published and preliminary data support the scientific premise that mineralocorticoid receptor (MR) antagonists, which have clinical benefit for DMD cardiomyopathy, stabilize muscle membranes, improve skeletal muscle force, and reduce fibrosis, are also anti-inflammatory and represent an ideal drug for combination with gene transfer. However, the anti- inflammatory properties of MR antagonists in muscular dystrophy have not been explored. Since prednisone directly competes with MR antagonist binding to its receptors, these studies are crucial for clinical use of MR antagonists as an adjunct therapy to replace prednisone. In this application, we will test whether prednisone and MR antagonists have the same or different effects on specific immune cell populations in dystrophic muscles, whether cytokine reductions by MR antagonists are dependent on MR signaling mechanisms in muscle fibers or myeloid cells, and whether MR antagonists limit accumulated damage after acute injury in dystrophic mice treated with micro-dystrophin gene therapy. We have developed methods to flow sort immune cell populations from single muscles from the mdx genotypic model of DMD that will allow the first identification of the immune cell populations suppressed by prednisone, despite decades of clinical use, and a direct comparison with MR antagonists. These methods will also allow for the unbiased identification of gene expression changes in inflammatory myeloid cells induced by MR antagonists and prednisone. These studies will inform optimal MR antagonist clinical use as a co-therapy to extend efficacy of emerging genetic therapies for DMD and potentially other forms of muscular dystrophies. The data generated will also identify novel potential anti-inflammatory treatment targets.