Three-model platform for understanding DMD epigenetic mechanisms and advancing small molecule therapies

项目总结 Duchenne肌营养不良症(DMD)是一种由DMD基因突变引起的严重退行性肌肉疾病 基因，它编码营养不良蛋白。目前的治疗标准是皮质类固醇治疗，延缓了病情的发展 肌肉功能障碍，但有严重的副作用。DMD基因治疗和基因编辑的方法是 前景看好，但面临着许多挑战。正在确定可能有益于DMD的药理方法 通过调节独立于肌营养不良蛋白突变的病理机制。然而，许多小分子 在通过DMD临床试验的过程中，治疗方法难以显示疗效，可能是由于 临床前评估不足或疾病进程中的靶向机制太晚。尽管DMD是 作为一种进展性疾病，有证据表明早期、胚胎和胎儿阶段的肌发生和基因缺陷 在DMD中的表达。特别是，我们的初步研究确定了已知的最早的DMD之一 表型：一种新的DMD人诱导多能干细胞(HiPSCs)经历 肌肉发生。通过了解这些早期的肌源性和转录缺陷是如何启动的，以及它们是如何驱动 DMD病理，我们可能更好地识别和利用DMD疗法。这是一个假设 建议是表观遗传药物，靶向染色质修饰和转录的小分子 调控，可以改善早期DMD转录缺陷以及改善下游的DMD病理。 某些组蛋白脱乙酰酶抑制剂(HDACi)在小鼠、斑马鱼和最近的DMD中显示出前景 临床试验。然而，除了这些hdaci外，表观遗传药物还没有得到广泛和系统的研究。 治疗DMD的有效性和安全性。这项提议的目标是识别新的表观遗传学小分子 通过在多个DMD模型中展示有效性而有益于DMD的分子，并且，并行地， 为了更好地描述DMD背后的转录和表观遗传机制。我们建议 三个目标。首先，我们将通过以下方式确定改善DMD表型的表观遗传小分子的类别 DMD斑马鱼表观遗传药库的筛选。我们的初步研究已经确定了小说 拯救DMD斑马鱼的表观遗传药物。第二，通过对HiPSC来源的DMD的单细胞基因组学研究 骨骼肌，我们将在启动过程中生成转录组和染色质包装缺陷的图谱 和DMD的进展。我们将测试表观遗传药物是否纠正了新的转录失调 我们已经在HiPSC来源的DMD肌肉中发现了表型和功能缺陷。第三，我们将 评价表观遗传药物在DMD大鼠中的有效性和安全性，评估临床相关功能 骨骼肌和心肌的转归。该项目将为早期的 在DMD中发生表观遗传失调。长期的影响将是发展三种模式 平台、斑马鱼、大鼠和HiPSC系，用于DMD药物发现和机制和功能验证， 利用每个模型系统的优势。

PROJECT SUMMARY Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by mutations in the DMD gene, which encodes dystrophin. The current standard of care, corticosteroid treatment, delays the progression of muscle dysfunction but has serious side effects. DMD gene therapy and gene editing approaches are promising but face many challenges. Pharmacological approaches are being identified that could benefit DMD by modulating pathological mechanisms independent of the dystrophin mutation. However, many small molecule therapies have had difficulties showing efficacy while progressing through DMD clinical trials, possibly due to inadequate preclinical evaluation or to targeting mechanisms too late in the disease process. Although DMD is a progressive disease, there is evidence for early, embryonic- and fetal-stage defects in myogenesis and gene expression in DMD. In particular, our preliminary studies have identified one of the earliest known DMD phenotypes: a novel transcriptional trajectory of DMD human induced pluripotent stem cells (hiPSCs) undergoing myogenesis. By understanding how these early myogenic and transcriptional defects initiate and how they drive DMD pathology, we may be better positioned to identify and utilize DMD therapies. The hypothesis of this proposal is that epigenetic drugs, small molecules that target chromatin modifications and transcriptional regulation, can ameliorate early DMD transcriptional defects as well as improve downstream DMD pathology. Certain histone deacetylase inhibitors (HDACi) have shown promise for DMD in mice, zebrafish, and recent clinical trials. However, beyond these HDACi, epigenetic drugs have not been broadly and systematically studied for their efficacy and safety to treat DMD. The goals of this proposal are to identify novel epigenetic small molecules that are beneficial for DMD, by demonstrating effectiveness in multiple DMD models, and, in parallel, to better characterize the disrupted transcriptional and epigenetic mechanisms underlying DMD. We propose three Aims. First, we will identify the classes of epigenetic small molecules that improve the DMD phenotype, by screening an epigenetic drug library in dmd zebrafish. Our preliminary studies have already identified novel epigenetic drugs that rescue dmd zebrafish. Second, through single-cell genomics on hiPSC-derived DMD skeletal muscle, we will generate maps of transcriptome and chromatin packaging defects during the initiation and progression of DMD. We will test whether epigenetic drugs correct the novel transcriptional dysregulation phenotype, as well as functional deficits, that we have identified in hiPSC-derived DMD muscle. Third, we will evaluate epigenetic drugs for efficacy and safety in the DMD rat, assessing clinically relevant functional outcomes in skeletal and cardiac muscle. This project will provide novel basic scientific insight into the early epigenetic dysregulation occurring in DMD. The long-term impact will be the development of a three-model platform, zebrafish, rat and hiPSC lines, for DMD drug discovery and mechanistic and functional validation, taking advantage of each model system.