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

• 批准号: 8952034
• 负责人: CHARLES A THORNTON
• 金额: $ 23.03万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8952034
• 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; 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; Relative (related person); 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; Tissues; Toxic effect; Transcript; Work; analytical method; base; clinical practice; cohort; experience; functional disability; functional improvement; gain of function; gel electrophoresis; minimally invasive; mouse model; mutant; next generation sequencing; novel; public health relevance; response; small molecule; success; targeted sequencing; targeted treatment; therapeutic target; therapy design; tibialis anterior muscle; tool; transcriptome sequencing; uptake

 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。遗传损伤是DMPK中CTG重复序列的扩增，其机制涉及含有扩增CUG重复序列（CUGexp）的转录本的毒性功能获得。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。很快，负责RNA毒性的治疗靶点被鉴定出来， 开发了作用于靶点的药物，并在小鼠模型中实现了至少在其早期阶段的疾病逆转。随着新的推定疗法进入临床试验，需要使用治疗反应的生物标志物尽早获得支持或反对靶点接合的确凿证据。该提案的目标是为此目的开发检测方法和生物标志物。主要重点放在选择性剪接，因为以前的工作表明，剪接失调是RNA毒性下游效应的最佳总体读数。此外，评估剪接调节的分析方法非常精确，并且在小鼠模型中进行的靶向治疗研究表明，剪接缺陷是完全可逆的，并且与功能改善密切相关。基于这些观察结果，以及从几个大型数据集中全面确定DM1相关剪接缺陷，我们在目标1中提出开发多重选择性剪接测序（MASseq），这是一种靶向高通量测序方法，用于评估小肌肉活检样本中的剪接生物标志物。为了使MASseq有资格用于生物标志物分析，我们将优化该方法，然后系统地检查采样方差、运行内和运行间的测定方差以及重测可靠性。后一项研究将使用来自相同受试者的成对肌肉样本，间隔八周收集。为了测试保真度和评估偏倚，将MASseq结果与来自相同样品的常规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毒性RNA细胞水平的治疗。我们将通过确定突变体与野生型转录本的比例，开发定量DMPK等位基因表达的方法。我们还将优化一种新的方法来测量CUGexp RNA的总细胞负荷。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，使用的方法可以很容易地转移到其他实验室。