Modulation of Jagged1/Pitpna in DMD as a means of therapy

项目摘要/摘要 Duchenne肌营养不良症(DMD)是一种退行性肌肉萎缩疾病，由基因突变引起 抗肌营养不良蛋白基因。肌萎缩蛋白在肌肉肌膜上的缺失导致肌肉数量增加。 对收缩引起的损伤以及二级信号通路的调节失调的敏感性，许多 其中的一些问题仍然知之甚少。尽管正在调查的治疗战略取得了进展，目的是 Dystrophin表达的恢复，包括微小dystrophin的病毒传递，翻译停止的通读 密码子、外显子跳过恢复阅读框，目前还没有治愈DMD的方法，而鉴定 独立于肌营养不良蛋白改善病理的治疗方法对患者将具有重要价值。在这 Vain，DMD的遗传修饰物正在成为潜在的治疗靶点。我们的实验室最近鉴定出 Jagged1和Pitpna作为金犬肌营养不良症DMD病理的遗传修饰物 (GRMD)犬群，其中两只特殊的“逃逸”犬表现出比 典型的GRMD狗，尽管缺乏dystrophin。正常情况下，GRMD犬表现出严重的表型 与人类DMD相似，包括早期进行性肌肉退化、纤维化和经常过早死亡 在生命的头两年内，由于心肺衰竭。逃生犬的基因表达分析 与严重感染GRMD的狗和对照动物相比，Jagged1过表达(OE) 和脑垂体表达降低是轻度表型的特征，包括 行走和正常寿命。在随后对营养不良蛋白缺陷型斑马鱼的遗传研究中，我们 证明Jagged1和Pitpna的调节可以防止营养不良肌肉的表现 表型，增加长期存活率，并改善游泳成绩。在原代成肌细胞中 正常和DMD患者，我们还显示Jagged1的过度表达和垂体抑制的影响 AKT/PTEN信号转导和促进成肌细胞融合。鉴于这些积极的数据，我们现在将扩大我们的研究范围 将Jagged1和Pitpna调制到特征良好的mdx5Cv小鼠DMD模型中。我们还将 研究PDE10A的遗传和药理抑制作用，我们已经证明PDE10A可以引起垂体肌的减少 营养不良蛋白缺陷型斑马鱼的表达和改善营养不良病理。这其中的长期目标是 该项目是评估和验证遗传修饰剂Jagged1和Pitpna的治疗潜力 改善DMD的病理，并寻找一种可行的药物调节剂进入临床研究。 为此，我们将按照以下三个具体目标进行实验：1) 2)研究mdx5Cv小鼠营养不良病理过程中，脑垂体性核糖核酸表达降低的影响。 Jagged1在mdx5Cv小鼠中过表达的功能作用；3)评估PDE10A的治疗潜力 抑制原代人类肌肉细胞、斑马鱼和DMD小鼠模型的营养不良病理。

PROJECT SUMMARY/ABSTRACT Duchenne Muscular Dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. The absence of dystrophin protein at the muscle sarcolemma results in increased muscle susceptibility to contraction-induced damage as well as dysregulation of secondary signaling pathways, many of which remain poorly understood. Despite advances in treatment strategies under investigation aimed at restoration of dystrophin expression including viral delivery of mini-dystrophin, read-through of translation stop codons, and exon skipping to restore the reading frame, there is no cure for DMD, and the identification of therapies that improve pathology independent of dystrophin would be of significant value to patients. In this vain, genetic modifiers of DMD are emerging as potential therapeutic targets. Our lab recently identified Jagged1 and Pitpna as genetic modifiers of DMD pathology in a Golden Retriever Muscular Dystrophy (GRMD) dog colony in which two exceptional “escaper” dogs exhibited a drastically milder phenotype than typical GRMD dogs despite being dystrophin-deficient. Normally, GRMD dogs show a severe phenotype similar to human DMD including early progressive muscle degeneration, fibrosis, and premature death often within the first 2 years of life due to cardiopulmonary failure. Gene expression analyses of the escaper dogs compared to severely affected GRMD dogs and control animals revealed that Jagged1 overexpression (OE) and decreased Pitpna expression were hallmarks of the mild phenotype, which including maintained ambulation and normal lifespan. In subsequent genetic studies of dystrophin-deficient zebrafish, we demonstrated that modulation of Jagged1 and Pitpna prevents manifestation of the dystrophic muscle phenotype, increases long-term survival, and improves swim performance. In primary myoblasts derived from normal and DMD patients, we also showed that Jagged1 overexpression and Pitpna inhibition impact AKT/PTEN signaling and improve myoblast fusion. Given this positive data, we will now extend our research of Jagged1 and Pitpna modulation into the well-characterized mdx5cv mouse model of DMD. We will also investigate genetic and pharmacological inhibition of PDE10A, which we have shown to elicit decreased Pitpna expression and improve dystrophic pathology in dystrophin-deficient zebrafish. The long-term goal in this project is to assess and validate the therapeutic potential of the genetic modifiers Jagged1 and Pitpna to ameliorate DMD pathology, and to identify a viable pharmacologic modulator to advance into clinical studies. To accomplish this, we will implement experiments in accordance to the following three specific aims: 1) Determine the effect of reduced Pitpna expression on dystrophic pathology in mdx5cv mice, 2) Characterize the functional role of Jagged1 overexpression in mdx5cv mice, and 3) Assess the therapeutic potential of PDE10A inhibition on dystrophic pathology in primary human muscle cells, zebrafish, and mouse models of DMD.