Pharmacodynamic Biomarker of Myotonic Dystrophy

抽象的。强直性肌营养不良1型(DM1)和2型(DM2)主要为遗传性退行性 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，一种 新的功能增益机制，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质，具有高亲和力 对于CUG-和CCUG-重复，会被重复的RNA捕获，导致转录产物的错误调控剪接 编码骨骼肌的关键成分。在小鼠模型中，我们使用了各种方法， 包括寡核苷酸、基因治疗载体和小分子，以表明RNA毒性的降低 导致RNA剪接和肌肉功能正常化，表明DM至少是部分可逆的。我们 还报道说，对错误调节的剪接事件进行靶向RNAseq分析对于 监测小鼠的治疗效果，提供对目标参与的近乎实时的评估。这些 发展和进行生物标记物驱动试验的前景刺激了许多药物的开发。 DM的计划，包括一些现在正在进行IND使能研究或早期试验的计划。至 为了准备这些研究，我们开发了类似于小鼠研究的人靶向RNAseq。 基于Deep 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本，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，我们为每个样本确定了一个综合剪接指数。拼接 指数对DM1患者的大队列中的人群进行分层，与肌肉无力的程度相关， 在2-3个月内显示可接受的拼接缺陷测试-重新测试协议，并提供可服务的 药效生物标志物。然而，随着领域的进步和我们获得更多的经验，重要的是 已经出现了局限性，特别是在获取肌肉样本的方法和拼接的选择方面 针对目标RNAseq的事件-紧密联系的两个方面。在这里，我们寻求优化并展示可行性 用于一种新的采样方法，称为肌抽吸法，它可以实现多次采样。即对更多肌肉进行采样 在更多的场合。同时，我们寻求优化和验证一种新的靶向RNAseq分析，适用于 DM1和DM2，专为容纳肌吸样本而设计，并提供更高的精度和 关于肌肉纤维中目标参与的更有力的推论。化验的性能最初将是 使用之前在我们的生物库中收集的样本进行评估，然后进行前瞻性评估 新收集的DM1和DM2患者的肌抽吸液样本，重点关注选择性地 受影响的或相对幸免的。建成后，预计该项目将提供一个简单而有效的 用于导致药物批准的治疗试验和用于批准后研究的药效生物标记物 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.