Mechanisms of mineralocorticoid receptor antagonism on inflammation in muscular dystrophy

项目总结 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 病人。然而，腺相关病毒(AAV)递送系统的局限性，包括其携带的小 肌肉干细胞的能力及其低效率的转基因，仍将是治愈的障碍。这个 基于Becker肌营养不良症的微型化转基因正在交付 肌营养不良蛋白)仍会导致一些骨骼肌周转，并伴随随后的炎症和心肌病。 此外，由于正常使用肌肉造成的损伤将被肌肉干细胞修复，而肌肉干细胞很可能无法修复 表达肌营养不良蛋白。这两个问题都会导致至少低水平的慢性炎症，这很可能 加剧肌肉损伤和转基因表达的最终丧失，限制疗效。强的松，它有 作为治疗DMD的标准，但有许多严重的副作用，继续作为一种抗 发炎，以防止对转基因的免疫反应。公布的和初步的数据支持 对DMD有临床益处的盐皮质激素受体(MR)拮抗剂的科学前提 心肌病，稳定肌膜，改善骨骼肌力，减少纤维化，也是 抗炎，是与基因转移相结合的理想药物。然而，反- MR拮抗剂在肌营养不良症中的炎症特性尚未被探索。由于泼尼松 与其受体结合的MR拮抗剂直接竞争，这些研究对MR的临床应用至关重要 作为替代强的松的辅助治疗的拮抗剂。在本申请中，我们将测试泼尼松是否 MR拮抗剂对营养不良患者特定免疫细胞群有相同或不同的影响 肌肉，MR拮抗剂是否减少细胞因子依赖于MR信号传导机制 肌纤维或髓系细胞，以及MR拮抗剂是否限制急性损伤后的累积损伤 用微营养不良蛋白基因治疗营养不良小鼠。我们已经开发出流动排序免疫的方法 来自DMD MDX基因分型模型的单个肌肉的细胞群将允许首次鉴定 强的松抑制的免疫细胞群体，尽管几十年的临床使用，和直接的 与MR对手进行比较。这些方法也将允许无偏见地识别基因。 MR拮抗剂和泼尼松对炎性髓系细胞表达的影响。这些研究 将提供最佳MR拮抗剂的临床应用，作为一种联合治疗，以延长新兴基因疗法的疗效 用于DMD和潜在的其他形式的肌肉营养不良。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.