Nanopore-based sequencing of placenta-cell-type-specific extracellular RNA for real time assessment of human placenta development and function

基于纳米孔的胎盘细胞类型特异性胞外 RNA 测序，用于实时评估人胎盘发育和功能

• 批准号: 10634502
• 负责人: Zev Williams
• 金额: $ 63.44万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10634502
• 关键词: Atlases; Biological Assay; Biological Markers; Blood; Cells; Characteristics; Circulation; Clinical; Code; Complementary DNA; Complex; Custom; DNA; Data; Decidua; Development; Diagnostic; Disease; Early Intervention; Exclusion; Fetal Growth; Fetal Growth Retardation; First Pregnancy Trimester; Functional disorder; Generations; Genes; Genetic Polymorphism; Grant; Hour; Human; Inflammatory; Information Protection; Institution; Learning; Libraries; Malignant Neoplasms; Measures; Messenger RNA; Metabolic; Methods; Mining; Monitor; Mothers; Nucleotides; Organ; Pathogenesis; Perfusion; Physicians; Physiology; Placenta; Plasma; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Premature Birth; Preparation; Protocols documentation; Publishing; RNA; Recovery; Reference Values; Research; Research Personnel; Sampling; Secure; Specific qualifier value; Speed; Structure; Supercomputing; Technology; Time; Tissues; Transcript; Translating; Validation; Villus; Whole Blood; cDNA Library; cell type; clinical application; cost; data warehouse; design; experience; extracellular; healthy pregnancy; in vivo monitoring; innovation; innovative technologies; insight; multidisciplinary; nanopore; new technology; next generation; novel; nuclease; point of care; preservation; prevent; real time monitoring; risk stratification; screening panel; stillbirth; targeted sequencing; tool; transcriptome sequencing

Circulating placenta-derived extracellular RNAs (exRNAs) offer tremendous potential as safe, highly-informative, non-invasive biomarkers for placental function because they provide dynamic data regarding the functional, metabolic, inflammatory and perfusion state of the placenta and reflect the status of the placenta at a cellular level beginning in the first trimester. Thus, they can reflect problems before structural or end-organ effects have occurred and thereby enable risk-stratification, early intervention, and provide valuable insights into disease pathogenesis. However, widespread clinical application of exRNAs as biomarkers for placenta function has been limited by numerous technical limitations: (1) Existing exRNA isolation kits/protocols have poor recovery rates, size biases, do not inactivate the ubiquitous nucleases sufficiently to preserve the information- rich long coding transcripts, and are too costly and not amenable to automated application that would be required in a clinical setting, (2) placenta-derived exRNAs represent only a fraction of total exRNAs and hence sequencing depth has to be very deep in order for them to be adequately sampled, (3) the full repertoire of placental cell types and characteristic transcripts were not known and thus could not be fully queried and, (4) existing sequencing technologies have library preparation and sequencing times that are too long to permit turn-around- times with sufficient speed to provide results in a timely and actionable manner. In this grant, we assemble a multidisciplinary and multi-institutional team to develop four innovative new technologies that we pioneered that, when combined and optimized, offer an opportunity to solve these limitations and make exRNA a transformative clinical and research tool. In Aim 1, we use Automated exRNA isolation (AxRI), a novel method for automated, high-throughput, low cost, isolation of exRNA with near-instantaneous inactivation of nucleases and consequent protection of the information-rich coding RNA transcripts to isolate exRNAs from maternal circulation. We then use a customized capture assay, adapted from one we developed for cancer panel screening, to enrich placenta-derived transcripts using transcript information from our single-cell atlas of the human placenta. In Aim 2, we further develop our nanopore-based library preparation and sequencing technology to sequence the transcripts on a hand-held, disposable nanopore-based DNA sequencer at speeds that are much faster than existing next-generation technologies and that can deliver same-day results. In Aim 3, we apply the technologies developed in Aims 1 and 2 to generate and validate reference profiles for exRNA transcripts across gestation beginning in the early first trimester. Collectively, the development, optimization, validation, and combination of the technologies proposed in this grant will result in a clinically-viable and affordable platform that will provide an unprecedented ability to safely and non-invasively assess placenta function and composition at a near-cellular level and transform how we monitor and study pregnancy.

循环胎盘来源的细胞外RNA（exRNA）提供了巨大的潜力，作为安全的，高度信息化的， 胎盘功能的非侵入性生物标志物，因为它们提供关于功能的动态数据， 胎盘的代谢、炎症和灌注状态，并反映胎盘在细胞内的状态。 从第一个三个月开始。因此，它们可以在结构或终末器官效应发生之前反映问题。 发生，从而使风险分层，早期干预，并提供有价值的见解，疾病 发病机制然而，exRNA作为胎盘功能的生物标志物的广泛临床应用 受到许多技术限制的限制：（1）现有的exRNA分离试剂盒/方案具有差的 回收率，大小偏差，不足以保护无处不在的核酸酶的信息- 丰富的长编码转录本，并且成本太高，并且不适合所需的自动化应用程序 在临床环境中，（2）胎盘来源的exRNA仅代表总exRNA的一部分，因此测序 深度必须非常深，以便充分采样，（3）胎盘细胞的全部剧目 类型和特征转录本未知，因此无法完全查询，（4）现有 测序技术具有太长的文库制备和测序时间而不允许周转， 以足够的速度提供及时和可操作的结果。在这份资助中，我们收集了一个 一个多学科和多机构的团队，开发四个创新的新技术，我们 开创性的，当组合和优化，提供了一个机会，以解决这些限制，使 exRNA是一种变革性的临床和研究工具。在目标1中，我们使用自动exRNA分离（AxRI）， 用于自动化、高通量、低成本、分离exRNA并具有近瞬时灭活的新方法 的核酸酶和随之而来的保护信息丰富的编码RNA转录本，以分离exRNA从 母体循环然后，我们使用定制的捕获分析，改编自我们为癌症小组开发的捕获分析。 筛选，以丰富胎盘来源的转录本使用转录本信息，从我们的单细胞图谱的 人胎盘在目标2中，我们进一步开发了基于纳米孔的文库制备和测序， 一种在手持式、一次性纳米孔DNA测序仪上对转录本进行快速测序的技术 比现有的下一代技术快得多，并且可以在同一天内提供结果。在目标3中， 我们应用目标1和2中开发的技术来生成和验证exRNA的参考图谱 从怀孕早期开始的转录。总的来说，开发，优化， 验证，并在此授权中提出的技术组合将导致临床可行的 一个经济实惠的平台，将提供前所未有的安全和非侵入性评估能力， 胎盘功能和组成在近细胞水平，并改变我们如何监测和研究 怀孕