Non-Invasive Prenatal Diagnostics Based on Circulating Trophoblasts

项目摘要/摘要 修订后的U01提案的长期目标是进行高级开发和严格的验证 一种新的基于循环滋养细胞(CTB)的无创性产前诊断(NIPD)技术，能够 一)从母体血液中富集/计数四氯化碳，以及二)分离单个四氯化碳，用于全基因组检测 妊娠前三个月的胎儿基因异常。另一项研究计划也是 提出用同样的工作流程分离和鉴定宫颈滋养细胞(TBS) 样本。 在母血中潜在的循环中的胎儿有核细胞(CFNC)中，CTB是一个理想的靶点 考虑到它们(I)寿命短，这排除了先前怀孕或 流产，(Ii)胎儿核型和基因型的表现，以及(Iii)独特的 既可用于浓缩又可用于鉴定的生物标志物。然而，分离纯净的四氯化碳一直是 由于其丰度极低，在技术上具有挑战性。 在过去的十年里，曾博士在加州大学洛杉矶分校的研究团队开发了嵌入纳米材料的 诊断平台(也称为NanoVelcro芯片)。为了开发纳米尼龙搭扣芯片的NIPD实用程序，该团队首先 开发了一种纳米压痕制造工艺，以制备与激光捕获显微解剖(LCM)兼容的 以经济高效和可扩展的方式制备纳米基质。这些芯片，结合使用捕获和 免疫细胞化学(ICC)试剂表现出卓越的CTB捕获性能。并行、高分辨率 已开发了显微成像和分析软件，以识别和注册 通过LCM实现对单个CTB的高精度分离。在与皮萨斯卡博士的合作下， 联合团队演示了从血液处理、单个CTB分离和DNA扩增开始的工作流程， 一直通过阵列CGH和/或下一代测序对CTB进行全基因组图谱分析。 我们的中心假设是，可以从5毫升的母血中提取10个四氯联苯(&gt；50 TB)。 宫颈样本)，从怀孕前三个月(8-12周)的孕妇采集 胎龄)，这些CTB/TBS的全基因组图谱可用于诊断胎儿遗传学 异常现象。在五年的资助期内，拟议的研究将通过两个具体的 目标：i)开发、优化和验证提出的基于CTB的NIPD技术，以及ii)进行初步 从加州大学洛杉矶分校和CSMC招募的孕妇的临床验证。联合小组设想， 建议的基于CTBS的NIPD技术的成功演示将引入一种革命性的NIPD 解决方案具有黄金标准诊断测试的敏感度和特异度，而不存在相关风险 胎儿。

PROJECT SUMMARY/ABSTRACT The long-term goal of this revised U01 proposal is to conduct advanced development and rigorous validation of an emerging circulating trophoblast (cTB)-based noninvasive prenatal diagnostic (NIPD) technology, capable of i) enriching/counting cTBs from maternal blood, and ii) isolating single cTBs for genome-wide detection of fetal genetic abnormalities during the first trimester of pregnancy. An alternative research plan is also presented to explore the use of the same workflow for isolating and characterizing trophoblasts (TBs) in cervix samples. Among potential circulating fetal nucleated cells (CFNCs) in maternal blood, cTBs are an ideal target considering their (i) short lifespan, which excludes the presence of cTBs from prior pregnancies or miscarriages, (ii) representation of fetal karyotype and genotype, and (iii) expression of a unique collection of biomarkers that can be used for both enrichment and identification. However, isolating pure cTBs has been technically challenging due to their extremely low abundance. Over the past decade, Dr. Tseng’s research team at UCLA has developed nanomaterial-embedded diagnostic platforms (a.k.a., NanoVelcro Chips). To exploit the NIPD utility of NanoVelcro Chips, the team first developed a nanoimprinting fabrication process to prepare the laser capture microdissection (LCM)-compatible nanosubstrates in a cost-efficient and scalable manner. These chips, in conjunction with the use of capture and immunocytochemistry (ICC) agents, exhibit superb cTB capture performance. In parallel, high-resolution microscopy imaging and analysis software has been developed to identify and register individual cTBs on the substrates, enabling highly accurate isolation of single cTBs by LCM. In collaboration with Dr. Pisarska, the joint team demonstrated a workflow starting with blood processing, single cTB isolation, and DNA amplification, all the way through whole genome profiling of cTBs by ArrayCGH and/or next generation sequencing. Our central hypothesis is that >10 cTBs can be harvested from 5-mL of maternal blood (>50 TBs from a cervix sample), collected from a pregnant woman during the first trimester of pregnancy (8-12 weeks of gestational age), and whole genome profiling of these cTBs/TBs can be used for diagnosing fetal genetic abnormalities. Over the 5-year funding period, the proposed research will be implemented via two Specific Aims: i) to develop, optimize and validate the proposed cTB-based NIPD technology, and ii) to conduct initial clinical validation in pregnant women recruited from UCLA and CSMC. The joint team envisions that the successful demonstration of the proposed cTBs-based NIPD technology will introduce a revolutionary NIPD solution with the sensitivity and specificity of the gold standard diagnostic tests without the associated risks to the fetus.