Circulating Organ-enriched microRNAs as biomarkers of Rett Syndrome

总结 Rett综合征（RTT）是一种罕见的X连锁神经发育障碍，由MECP2基因突变引起。 这种疾病几乎只影响女性，因为MECP2突变的男性胎儿通常在出生前死亡。 出生受影响的女性在出生时表现正常;疾病发作通常在6 - 18个月内明显， 其特征在于神经退化、运动刻板、不规则呼吸和其他障碍。临床 严重程度似乎取决于多种因素，包括MECP2突变的类型，X- MECP 2的失活和可能的遗传修饰剂。虽然MECP2基因检测可用于 RTT，RTT的外周生物标志物，包括疾病严重程度的微创、基于血液的指标， 进步，缺乏。分子诊断公司DiamiR开发了专有平台 基于循环分析的病理生理过程的早期检测和监测技术 器官富集的，包括脑富集的，血浆中的microRNA（miRNAs）。迄今为止， 该公司鉴定了有前途的miRNA生物标志物候选物，用于神经退行性疾病和其他疾病。 疾病，在这里，我们建议扩展我们的技术的使用，以建立RTT的miRNA生物标志物。 MECP2的缺失导致突触功能障碍和脑中miRNA的显著失调。我们 假设循环中的miRNAs在不同的脑区富集，存在于突触/神经突中， 在血浆中可检测的蛋白质可以检测与RTT发展相关的病理生理过程。此外，本发明还提供了一种方法， 分析肝脏、肺和肌肉中富集的miRNA水平可以反映RTT相关的变化， 这些组织。使用"miRNA对"方法进行数据标准化，并且包含生物标志物候选物。 两种miRNAs的比例。将两到三个miRNA对组合到miRNA分类器中以获得更高的准确性。 用四种小鼠模型和人类患者进行的初步研究鉴定了miRNA对和分类器 区分RTT小鼠模型与野生型小鼠以及RTT患者与年龄匹配的对照（AMC）; 某些miRNA对在小鼠和人类中是共同的，表明潜在的miRNA之间的相似性。 病理过程。本研究将使用在Rett中心采集的血浆样本进行 在蒙特菲奥里医疗中心具体目标包括：（1）评估区分RTT和AMC的可行性 通过在受RTT影响的器官/组织（脑，肝， 肺、肌肉）（60 RTT/60 AMC）;和（2）评估循环miRNA用于预测疾病的潜力 通过分析先前研究的19种miRNAs的血浆水平， 3年后从同一研究参与者（30 RTT/30 AMC）收集的纵向样本组中。 SBIR II期将涉及更大规模的临床研究，以验证miRNA生物标志物候选物。对试剂盒进行 最初开发为临床试验测定，并且在积累临床数据后，开发为体外诊断。

SUMMARY Rett syndrome (RTT) is a rare X-linked neurodevelopmental disorder caused by mutations in the MECP2 gene. The disease affects females almost exclusively, since male fetuses with a MECP2 mutation usually die before birth. Affected females appear normal at birth; disease onset is typically evident by 6-18 months and is characterized by neurological regression, motor stereotypies, irregular breathing, and other handicaps. Clinical severity appears to depend on multiple factors, including the type of MECP2 mutation, skewing of the X- inactivation and likely genetic modifiers of MECP2. Although diagnostic MECP2 genetic testing is available for RTT, peripheral biomarkers of RTT, including minimally invasive, blood-based indicators of disease severity and progression, are lacking. DiamiR, a molecular diagnostics company, has developed proprietary platform technology for early detection and monitoring of pathophysiological processes based on analysis of circulating organ-enriched, including brain-enriched, microRNAs (miRNAs) in plasma. Studies conducted to date at the company resulted in identification of promising miRNA biomarker candidates for neurodegenerative and other diseases, and here we propose extending the use of our technology to establish miRNA biomarkers of RTT. Loss of MECP2 results in synaptic dysfunction and marked dysregulation of miRNAs in the brain. We hypothesize that circulating miRNAs enriched in different brain regions, present in synapses/neurites, and detectable in plasma can detect pathophysiological processes associated with RTT development. In addition, analysis of levels of miRNAs enriched in liver, lung and muscle can be reflective of RTT associated changes in these tissues. An “miRNA pair” approach is used for data normalization, and a biomarker candidate is comprised of a ratio of two miRNAs. Two-three miRNA pairs are combined into a miRNA classifier for higher accuracy. Preliminary studies performed with four murine models and human patients identified miRNA pairs and classifiers differentiating both RTT mouse models from wild-type mice and RTT patients from age-matched control (AMC); certain miRNA pairs were common to mice and humans, indicating the similarity between the underlying pathological processes. The current study will be performed using plasma samples collected at the Rett Center at Montefiore Medical Center. Specific aims include: (1) assessing the feasibility of differentiating RTT from AMC by plasma levels of 38 pre-selected circulating miRNAs enriched in organs/tissues affected by RTT (brain, liver, lung, muscle) (60 RTT/60 AMC); and (2) evaluating the potential of circulating miRNAs for predicting disease severity and monitoring disease progression by analysis of plasma levels of the previously studied 19 miRNAs in the longitudinal set of samples collected 3 years later from the same study participants (30 RTT/30 AMC). SBIR Phase II will involve larger clinical studies to validate miRNA biomarker candidates. The assay will be initially developed as a clinical trial assay, and, upon accumulation of clinical data, as in vitro diagnostics.