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

循环中的胎盘来源的细胞外RNA(ExRNA)提供了巨大的潜力，作为安全的，高度信息量， 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、炎症和灌流状态，并反映胎盘在细胞内的状态 从怀孕前三个月开始的水平。因此，它们可以在结构或末端器官效应发生之前反映问题。 发生，从而实现风险分层、早期干预并提供对疾病的有价值的见解 发病机制。然而，exRNAs作为胎盘功能的生物标志物在临床上的广泛应用 受到许多技术限制的限制：(1)现有的exRNA分离试剂盒/协议很差 回收率，大小偏差，不足以使普遍存在的核酸酶失活，以保存信息- 丰富的长编码抄本，成本太高，不适合所需的自动化应用 在临床环境中，(2)胎盘来源的exRNAs只占总exRNAs的一小部分，因此测序 深度必须非常深，才能充分采样；(3)胎盘细胞的全部谱系 类型和特征成绩单未知，因此无法完全查询，以及，(4)存在 测序技术的文库准备和测序时间太长，无法扭转局面-- 以足够的速度以及时和可操作的方式提供结果。在这笔赠款中，我们汇集了一个 多学科和多机构团队开发四项创新的新技术 开创性的，当组合和优化时，提供了解决这些限制的机会，并使 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)，即 一种自动化、高通量、低成本、近瞬时失活的外源RNA提取新方法 核酸酶和由此产生的信息丰富的编码RNA转录本的保护，以从 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分析 筛选，使用我们的单细胞图谱中的转录信息丰富胎盘来源的转录本 人类胎盘。在目标2中，我们进一步发展了基于纳米孔的文库制备和测序 在手持、一次性纳米孔DNA测序仪上快速对转录进行测序的技术 它比现有的下一代技术快得多，而且可以在同一天内交付结果。在《目标3》中， 我们应用AIMS 1和AIMS 2中开发的技术来生成和验证exRNA的参考配置文件 妊娠早期三个月开始的成绩单。总的来说，开发、优化、 这项资助中建议的技术的验证和组合将导致临床上可行的 经济实惠的平台，将提供前所未有的安全、非侵入性评估能力 近细胞水平的胎盘功能和组成，并改变我们监测和研究的方式 怀孕了。

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.