Placenta-derived extracellular circulating RNA as a tool for monitoring placental function

 描述(申请人提供)：胎盘是胎儿和母亲之间的纽带；胎盘健康绝对是胎儿和母亲健康的核心和关键。胎盘是胎儿营养和氧气的来源，允许胎儿排出废物，保护胎儿免受毒素、环境暴露、感染剂和母体免疫系统排斥的影响，并产生支持怀孕的激素。胎盘结构和功能的异常是妊娠相关并发症的基础，包括早产、流产和死产，以及先兆子痫等高血压疾病。因此，监测胎盘功能是必要的，以防止母亲和胎儿的不良后果。及早发现胎盘功能的变化为加强妊娠监测和治疗提供了机会。与其他所有器官一样，胎盘的功能状态是通过编码和非编码基因表达的变化来反映的。因此，监测胎盘转录组可以全面评估胎盘功能。挑战是能够在不使用侵入性方法(例如胎盘活检)的情况下评估胎盘转录组，因为这些方法对妊娠构成风险。为了具有临床相关性，这种方法必须是可靠的，能够以合理的成本用于大量临床样本，并能够监测完整的胎盘转录组。不幸的是，这些目标在现有技术下是不可能实现的。我们实验室已经建立了一种RNA测序方法来全面和定量地描述细胞外RNA(ExRNA)，特别是在母体循环中发现的胎盘microRNAs(MiRNAs)。在拟议的项目中，我们将推进这一方法，并开发能够通过母体血液和尿液对胎盘转录组进行非侵入性监测的技术。在目标1中，我们将开发两项新技术，自动exRNA分离(AxRI)和全短RNA测序(TsRNAseq)，这将使从母体血液和尿液中分离和测序编码和非编码exRNA成为可能。在目标2中，我们将应用这些新技术来监测足月和第一、中期妊娠孕妇血液和尿液中胎盘特异的exRNAs；这些较早的时间点很重要，因为这些时间点是转录组变化最具临床相关性和生物信息学意义的孕期。在目标3中，我们将定义胎盘特异性exRNAs的确切细胞来源，为分析exRNAs提供上下文信息。因此，该项目的规模提供了前所未有的机会，利用尖端技术进行exRNA分离和检测、测序和生物信息学，以开发一种非侵入性实时监测胎盘转录组的工具。然后，这一工具可用于深入了解妊娠相关疾病的发病机制，在终末器官损害发生之前确定高危妊娠，并最终提供治疗干预措施，以改善孕产妇和新生儿的健康。

 DESCRIPTION (provided by applicant): The placenta is the nexus between the fetus and mother; placental health is absolutely central and critical to both fetal and maternal health. The placenta is the source of nutrition and oxygenation for the fetus, allows the fetus to eliminate waste, protects it from toxins, environmental exposures, infectious agents and rejection by the maternal immune system, and produces hormones that support the pregnancy. Abnormalities in placental structure and function underlie complications associated with pregnancy, including preterm birth, miscarriage and stillbirth, and hypertensive disorders such as preeclampsia. Therefore, monitoring placental function is necessary in order to prevent adverse outcomes for the mother and the fetus. Early detection of changes in placental function provides the opportunity for increased pregnancy surveillance and treatment. As is the case with every other organ, the functional state of the placenta is reflected by changes in the expression of coding and non-coding genes. Thus, monitoring the transcriptome of the placenta allows for a comprehensive assessment of placental function. The challenge is to be able to assess placental transcriptome without using invasive methods (e.g. placental biopsy) because these methods pose a risk to the pregnancy. To be clinically relevant, this method must be robust, reliable and able to use on large numbers of clinical samples at reasonable costs, and be able to monitor the full placental transcriptome. Unfortunately, these goals are not possible with existing technology. Our laboratory has developed an RNA sequencing method to comprehensively and quantitatively profile extracellular RNA (exRNA), specifically, placental microRNAs (miRNAs), found in maternal circulation. In the proposed project, we will advance this method and develop technology that will enable non-invasive monitoring of the placental transcriptome through the maternal blood and urine. In Aim 1, we will develop two new technologies, automated exRNA Isolation (AxRI) and total short RNA sequencing (tsRNAseq) that will enable isolation and sequencing of coding and non-coding exRNAs from the maternal blood and urine. In Aim 2, we will apply these new technologies to monitor placenta-specific exRNAs in the maternal blood and urine of term and first and second trimester pregnancies; these earlier time points are important because these is the gestational ages when changes in the transcriptome will be the most clinically relevant and biologically informative. In Aim 3, we will define the precise cell-origin of placenta-specific exRNAs to provide contextual information to analysis of the exRNAs. Therefore, the scope of this project provides the unprecedented opportunity to leverage cutting-edge technologies for exRNA isolation and detection, sequencing, and bioinformatics, in order to develop a tool to non-invasively monitor the transcriptome of the placenta in real time. This tool can then be used to gain insights into pathogenesis of pregnancy-related diseases, identify high-risk pregnancies before the onset of end-organ damage and, ultimately, provide therapeutic interventions to improve maternal and neonatal health.