Prospective study in myotonic dystrophy to determine extracellular RNA biomarkers

总结 强直性肌营养不良（dystrophicamyotonica; DM）是一种遗传性神经退行性疾病， 进行性肌无力、认知功能障碍和睡眠障碍。没有有效的治疗方法 可以减缓或逆转症状。DM 1型（DM 1）是由DM中CTG重复序列扩增引起的。 蛋白激酶（DMPK）基因。临床特征是由DMPK mRNA的致病作用引起的， 至少几十个转录物的可变前mRNA剪接的失调。在临床前研究中， 肌肉组织中的选择性剪接结果已被用作 治疗性反义寡核苷酸（阿索）药物反应。许多DM 1患者也会出现中枢神经系统疾病。 睡眠呼吸暂停、白天过度嗜睡、工作记忆减退、视觉空间技能受损，以及 解决问题的能力不足。这些中枢神经系统（CNS）症状与错误- 调节的选择性剪接模式和基因表达的变化在死后的大脑。脑脊液 (CSF)包围并保护大脑和中枢神经系统免受损伤。脑脊液成分的变化可以 作为活体CNS分子病理学变化的早期指标。然而，方法 对于使用CSF测量CNS中DM 1特异性剪接模式或基因表达变化， 不可用.细胞外RNA（exRNA）是指mRNA和非编码RNA，其从细胞内释放， 作为一种新的细胞间通讯形式。我们最近的研究表明， 尿exRNA的结果有能力作为外周疾病活动的可靠生物标志物， DM1。在这项前瞻性研究中，我们将测试exRNA包含代表性转录组的假设， 在DM 1中改变并在CSF中可定量。CSF exRNA中的剪接结果预计与 使用CNS功能的临床测量与DM 1疾病活动性相关。目标1将确定和表征 DM 1患者和未受影响的对照受试者的CSF exRNA谱。液滴数字PCR（ddPCR），一种 最先进的技术，将用于定量一组CSF exRNA剪接事件，以前报告为改变 在DM 1 CNS中。为了鉴定新的潜在生物标志物，我们将进行exRNA测序。目标2将决定 在DM 1的前瞻性队列中通过纵向监测的exRNA谱的测试/再测试可靠性 患者目的3将建立exRNA谱和定量临床测量之间的关系， CNS疾病活动，包括认知测试和脑部MRI。拟议用途：监测 用于DM 1患者疾病状态和负担的系列评估的生物标志物。在未来的临床试验中，CSF exRNA生物标志物将使患者分层和分子疾病活动的方便监测成为可能 在药物治疗过程中的中枢神经系统。本建议中使用的方法也将适用于 2型糖尿病（DM 2）和其他神经退行性疾病更普遍。

Summary Myotonic dystrophy (dystrophia myotonica; DM) is an inherited neurodegenerative disease that causes progressive muscle weakness, cognitive dysfunction, and sleep disturbance. No effective treatments are available to slow or reverse symptoms. DM type 1 (DM1) is caused by an expanded CTG repeat in the DM protein kinase (DMPK) gene. Clinical features result from pathogenic effects of the DMPK mRNA that lead to deregulation of alternative pre-mRNA splicing of at least several dozen transcripts. In pre-clinical studies, alternative splicing outcomes in muscle tissue have been used as sensitive pharmacodynamic indicators of therapeutic antisense oligonucleotide (ASO) drug response. Many patients with DM1 also experience central sleep apnea, excessive daytime sleepiness, diminished working memory, impaired visuospatial skills, and deficits in problem-solving skills. These central nervous system (CNS) symptoms are associated with mis- regulated alternative splicing patterns and gene expression changes in post-mortem brain. Cerebrospinal fluid (CSF) surrounds and protects the brain and CNS from injury. Changes in the composition of CSF can serve as early indicators of changes in the molecular pathology of the CNS in living individuals. However, methods for the use of CSF to measure DM1-specific splicing patterns or gene expression changes in the CNS are unavailable. Extracellular RNA (exRNA) refers to mRNAs and noncoding RNAs that are released from and taken up by cells as a novel form of cell-to-cell communication. Our recent work suggests that splicing outcomes in urine exRNA have the capacity to serve as robust biomarkers of peripheral disease activity in DM1. In this prospective study, we will test the hypothesis that exRNA contains a representative transcriptome that is altered in DM1 and quantifiable in CSF. Splicing outcomes in CSF exRNA are expected to correlate with DM1 disease activity using clinical measures of CNS function. Aim 1 will identify and characterize the CSF exRNA profile in DM1 patients and unaffected control subjects. Droplet digital PCR (ddPCR), a state-of- the-art technology, will be used to quantify a panel of CSF exRNA splice events previously reported as altered in DM1 CNS. To identify new potential biomarkers, we will perform exRNA sequencing. Aim 2 will determine the test/re-test reliability of the exRNA profile by longitudinal monitoring in a prospective cohort of DM1 patients. Aim 3 will establish a relationship between the exRNA profile and quantitative clinical measures of CNS disease activity, including cognitive testing and brain MRI. Proposed context of use (COU): monitoring biomarker for the serial assessment of disease status and burden in DM1 patients. In future clinical trials, CSF exRNA biomarkers would enable patient stratification and convenient monitoring of molecular disease activity in the CNS during the course of drug treatment. The approach used in this proposal also will be applicable to DM type 2 (DM2) and to other neurodegenerative diseases more generally.