Biomarkers of therapeutic response in myotonic dystrophy

强直性肌营养不良治疗反应的生物标志物

基本信息

批准号：
9098817
负责人：
CHARLES A THORNTON
金额：
$ 19.19万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9098817
关键词：
Alternative Splicing Antisense Oligonucleotides Biological Assay Biological Markers Biopsy Biopsy Specimen Capillary Electrophoresis Characteristics Clinical Research Clinical Trials Couples Data Data Set Decision Making Defect Development Disease Dose Event Genetic Goals Health High-Throughput Nucleotide Sequencing Laboratories Lesion Measurement Measures Messenger RNA Methods Muscle Muscle Fibers Muscle Weakness Muscular Dystrophies Mutation Myotonic Dystrophy Pathogenesis Patients Performance Pharmaceutical Preparations Phase Procedures Qualifying RNA RNA Splicing RNA-Directed DNA Polymerase Regulation Reporting Reverse Transcriptase Polymerase Chain Reaction Role Running Sampling Severities Severity of illness Single Nucleotide Polymorphism Staging Systematic Bias Testing Therapeutic Therapeutic Trials Tissue Sample Toxic effect Transcript Work analytical method base clinical practice cohort experience functional disability functional improvement gain of function gel electrophoresis knock-down minimally invasive mouse model mutant next generation sequencing novel small molecule success targeted sequencing targeted treatment therapeutic target therapy design tibialis anterior muscle tissue biomarkers tool transcriptome sequencing treatment response uptake

项目摘要

DESCRIPTION (provided by applicant): Myotonic dystrophy type 1 (DM1) differs from other forms of muscular dystrophy in having an unconventional mutation and a novel disease mechanism. The genetic lesion is an expanded CTG repeat in DMPK, and the mechanism involves a toxic gain-of-function by transcripts containing an expanded CUG repeat (CUGexp). Recent studies have suggested that these unique characteristics create a major therapeutic opportunity. In rapid succession, therapeutic targets responsible for RNA toxicity were identified, agents acting on targets were developed, and reversal of the disease, at least in its early stages, was accomplished in mouse models. As new putative therapies advance to clinical trials, there is a need to obtain conclusive evidence for or against target engagement, at the earliest possible moment, using biomarkers of therapeutic response. The goal of this proposal is to develop assays and biomarkers for this purpose. Major emphasis is placed on alternative splicing because previous work has suggested that splicing misregulation is the best overall readout for downstream effects of RNA toxicity. In addition, analytical methods to assess splicing regulation are very precise, and studies of targeted therapy in mouse models have shown that splicing defects are fully reversible and closely aligned with functional improvement. Building on these observations, and a comprehensive ascertainment of DM1-associated splicing defects from several large data sets, we propose in Aim 1 to develop Multiplex Alternative Splice sequencing (MASseq), a targeted high-throughput sequencing method to assess splicing biomarkers in small muscle biopsy samples. In an effort to qualify MASseq for biomarker analysis, we will optimize the method and then systematically examine sampling variance, assay variance within and between runs, and test-retest reliability. The latter studies will use paired muscle samples from the same subjects, collected eight weeks apart. To test fidelity and assess bias, MASseq results will be compared to reference data from conventional RNAseq of the same samples. Finally, associations of MASseq with functional impairment will be examined. While splicing biomarkers are applicable across all therapeutic strategies for DM1, our second Aim is specifically designed for treatments that reduce cellular levels of toxic RNA. We will develop methods to quantify allelic expression of DMPK, by determining the ratio of mutant versus wild-type transcripts. We will also optimize a novel method to measure the total cellular burden of CUGexp RNA. The results of this project will provide useful biomarkers to guide decision making in clinical trials and potentially in clinical practice, using methods that ae readily transferable to other laboratories.

描述（申请人提供）：强直性肌营养不良1型（DM1）与其他形式的肌营养不良的不同之处在于具有非常规突变和新的疾病机制，其遗传损伤是DMPK中CTG重复的扩展，其机制涉及毒性增益。最近的研究表明，这些独特的特征创造了一个重要的治疗机会，导致 RNA 毒性的治疗靶点被快速确定。随着新的假定疗法进入临床试验，需要获得支持或反对靶点参与的决定性证据，并且开发了作用于靶点的药物，并且至少在早期阶段已经在小鼠模型中实现了疾病的逆转。该提案的目标是开发用于此目的的检测方法和生物标记物，因为先前的工作表明剪接错误调节是下游效应的最佳整体读数。 RNA毒性。此外，评估剪接调节的分析方法非常精确，并且在小鼠模型中进行的靶向治疗研究表明，剪接缺陷是完全可逆的，并且基于这些观察结果以及对 DM1 相关剪接缺陷的全面确定，剪接缺陷与功能改善密切相关。根据多个大型数据集，我们在目标 1 中建议开发多重选择性剪接测序 (MASseq)，这是一种有针对性的高通量测序方法，用于评估小肌肉活检样本中的剪接生物标志物。为了使 MASseq 能够进行生物标志物分析，我们将优化该方法，然后配对系统地检查采样方差、运行内和运行之间的方差分析以及重测可靠性，后者的研究将使用来自同一受试者的肌肉样本，这些样本间隔八周收集。测试保真度并评估偏差，MASseq 结果将与相同样本的常规 RNAseq 的参考数据进行比较，最后，将检查 MASseq 与功能障碍的关联，而剪接生物标志物适用于 DM1 的所有治疗策略，这是我们的第二个目标。专为降低细胞毒性 RNA 水平的治疗而设计，我们将通过确定突变型与野生型转录本的比例来开发量化 DMPK 等位基因表达的方法。 CUGexp RNA。该项目的结果将提供有用的生物标志物，以指导临床试验和潜在的临床实践中的决策，使用易于转移到其他实验室的方法。