Pharmacodynamic Biomarker of Myotonic Dystrophy

摘要。强直性肌营养不良1型（DM1）和2型（DM2）是显性遗传的退行性疾病， 导致肌肉无力和多系统症状的疾病。这两种疾病都涉及RNA毒性， 新的功能获得机制，由具有数千个CUG或CCUG的RNA表达触发， 重复（分别为DM1或DM2）。调节RNA选择性剪接的关键蛋白，具有高亲和力 对于CUG-和CCUG-重复序列，被捕获在重复RNA上，导致转录本的错误调节剪接 编码骨骼肌的关键成分在小鼠模型中，我们使用了多种方法， 包括寡核苷酸，基因治疗载体和小分子，以显示RNA毒性的降低 导致RNA剪接和肌肉功能正常化，表明DM至少部分可逆。我们 也报道了错误调节的剪接事件的靶向RNAseq分析是可靠和敏感的， 监测小鼠中的治疗效果，提供靶点参与的近实时评估。这些 生物标志物驱动试验的发展和前景，刺激了许多药物的开发 包括一些正在推进IND研究或早期试验的项目。到 为了准备这些研究，我们开发了与小鼠研究中使用的类似的人靶向RNAseq。 基于深度RNAseq对90多个肌肉样本的全面发现，我们确定了 显示剪接失调的转录物，然后开发靶向RNAseq来评估22个剪接事件 在DM1中表现出很大的影响。然后，我们确定了每个样品的复合剪接指数。拼接 指数对DM 1患者的大队列中的人群进行分层，与肌无力的程度相关， 在2 - 3个月内显示拼接缺陷的可接受的测试-重测协议，并提供可用的 药效学生物标志物。然而，随着该领域的发展，我们获得了更多的经验，重要的是， 但也出现了局限性，特别是在获取肌肉样品的方法和剪接的选择方面。 针对靶向RNAseq的事件-这两个方面密切相关。在这里，我们寻求优化和展示可行性 一种新的采样方法，称为myoaspiration，可以进行多次采样。即更多肌肉的采样 在更多的场合。与此同时，我们寻求优化和验证一种新的靶向RNAseq检测方法，适用于 DM1和DM2，设计用于容纳肌抽吸物样本，并提供更高的精度， 对肌肉纤维中的目标参与的更强的推断。试验性能最初将 使用我们生物库中先前收集的样本进行评估，然后进行前瞻性评估， 从DM1和DM2患者中新收集的肌抽吸物样本，重点关注选择性 受影响或相对幸免。项目完成后，预计将提供一个简单有效的 用于导致药物批准的治疗试验和用于批准后研究的药效学生物标志物， 比较不同药物、方案和组合的有效性。

ABSTRACT. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominantly-inherited degenerative disorders that cause muscle weakness and multi-systemic symptoms. Both disorders involve RNA toxicity, a novel gain-of-function mechanism, triggered by expression of RNAs that have thousands of CUG- or CCUG- repeats (DM1 or DM2, respectively). Key proteins that regulate RNA alternative splicing, that have high affinity for CUG- and CCUG-repeats, become trapped on repetitive RNA, causing mis-regulated splicing of transcripts encoding critical components of skeletal muscle. In mouse models we have used a variety of approaches, including oligonucleotides, gene therapy vectors, and small molecules, to show that reduction of RNA toxicity causes normalization of RNA splicing and muscle function, suggesting that DM is at least partly reversible. We also reported that targeted RNAseq analysis of misregulated splice events is reliable and sensitive for monitoring therapeutic impact in mice, providing a near-real-time assessment of target engagement. These developments, and prospects for conducting biomarker-driven trials, have spurred many drug development programs for DM, including some that are now advancing to IND-enabling studies or early-phase trials. To prepare for these studies we developed human targeted RNAseq similar to that employed in mouse studies. Building on comprehensive discovery by deep RNAseq of more than 90 muscle samples, we identified transcripts that show splicing dysregulation, and then developed targeted RNAseq to evaluate 22 splice events that show large effects in DM1. We then determined a composite splicing index for each sample. The splicing index stratifies the population in a large cohort of DM1 patients, correlates with the extent of muscle weakness, shows acceptable test-retest agreement of splicing defects over 2-3 months, and provides a serviceable pharmacodynamic biomarker. However, as the field advances and we gain more experience, important limitations have emerged, particularly in the method for obtaining muscle samples and the selection of splice events for targeted RNAseq – two aspects that are closely linked. Here we seek to optimize and show feasibility for a new sampling method, called myoaspiration, that enables multi-sampling. i.e. sampling of more muscles on more occasions. In parallel, we seek to optimize and validate a new targeted RNAseq assay, applicable to both DM1 and DM2, designed to accommodate myoaspirate samples, and providing greater precision and stronger inferences about target engagement in muscle fibers. Performance of the assay will be initially evaluated using previously collected samples in our BioBank, and then proceed to prospective evaluation of newly-collected myoaspirate samples from DM1 and DM2 patients, focusing on the muscles that are selectively affected or relatively spared. Upon completion, the project is expected to provide a simple yet effective pharmacodynamic biomarker for therapeutic trials leading to drug approval, and for post-approval studies to compare the effectiveness of different agents, regimens, and combinations.