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（由适用提供）：1型肌发育症（DM1）与其他形式的肌肉营养不良不同，在具有非常规突变和新型疾病机制方面。遗传病变是DMPK中的CTG重复膨胀，该机制涉及通过包含扩展的CUG重复（CugeXP）的转录物获得的功能获得功能。最近的研究表明，这些独特的特征创造了主要的治疗机会。在快速连续的过程中，确定了负责RNA毒性的治疗靶标，开发了作用于靶标的药物，并且在小鼠模型中至少在早期阶段就逆转了疾病。随着新推定的疗法进行临床试验，需要使用治疗反应的生物标志物来获得最早或反对目标参与的证据。该提案的目的是为此目的开发评估和生物标志物。主要重点是替代剪接，因为先前的工作表明，剪接不调节是RNA毒性下游影响的最佳总读数。此外，评估剪接调节的分析方法非常精确，并且在小鼠模型中对靶向治疗的研究表明，剪接缺陷是完全可逆的，并且与功能改善紧密相符。在这些观察结果的基础上，以及来自几个大数据集的DM1相关剪接缺陷的全面确定，我们在AIM 1中提出，以开发多重替代剪接测序（MASSEQ），这是一种靶向的高通量测序方法，这是一种评估小型肌肉活检样本中生物标记物的启动性生物标志物。为了使MASSEQ有资格进行生物标志物分析，我们将优化该方法，然后系统地检查采样差异，运行和之间的测定方差以及测试可靠性。后来的研究将使用来自同一受试者的配对肌肉样本，相距八周。为了测试保真度和评估偏见，将将MASSEQ结果与来自相同样本的常规RNASEQ的参考数据进行比较。最后，将检查MASSEQ与功能障碍的关联。虽然剪接生物标志物适用于DM1的所有治疗策略，但我们的第二个目标是专门设计用于降低细胞毒性RNA水平的治疗方法。我们将通过确定突变体与野生型转录本的比率来开发量化DMPK的seartion表达的方法。我们还将优化一种新的方法来测量CugeXP RNA的总细胞伯宁。该项目的结果将提供有用的生物标志物，以使用AE容易转移到其他实验室的方法来指导临床试验中的决策，并有可能在临床实践中进行决策。