Non-Invasive Prenatal Diagnostics Based on Circulating Trophoblasts

项目总结/摘要 本修订U 01提案的长期目标是进行以下方面的高级开发和严格验证： 一种新兴的基于循环滋养层（cTB）的无创产前诊断（NIPD）技术，能够 i）富集/计数来自母体血液的cTB，和ii）分离单个cTB用于全基因组检测， 在怀孕的前三个月胎儿基因异常。另一项研究计划也是 提出了探索使用相同的工作流程分离和表征子宫颈滋养细胞（TB） 样品 在母体血液中潜在的循环胎儿有核细胞（CFNCs）中，cTB是理想的靶点 考虑到他们的（i）寿命短，排除了先前妊娠的cTB的存在，或 流产，（ii）胎儿核型和基因型的代表，和（iii）表达的独特集合， 生物标志物，可用于富集和鉴定。然而，分离纯的cTB一直是 技术上具有挑战性，因为它们的丰度极低。 在过去的十年里，曾博士在加州大学洛杉矶分校的研究小组已经开发出了纳米材料嵌入 诊断平台（也称为，NanoVelcro芯片）。为了利用NanoVelcro芯片的NIPD效用，该团队首先 开发了一种纳米压印制造工艺，以制备激光捕获显微切割（LCM）兼容 以成本有效和可扩展的方式制造纳米衬底。这些芯片结合捕获和 免疫细胞化学（ICC）试剂表现出极好的cTB捕获性能。并行，高分辨率 已经开发了显微成像和分析软件，以识别和登记细胞上的单个cTB。 在不同的基质上，能够通过LCM高度精确地分离单个cTB。与Pisarska博士合作， 联合团队展示了从血液处理、单个cTB分离和DNA扩增开始的工作流程， 通过ArrayCGH和/或下一代测序对cTB进行全基因组分析。 我们的中心假设是从5 mL母体血液中可以收获>10个cTB（从母体血液中可以收获>50个TB）。 子宫颈样品），在怀孕的前三个月（8-12周）期间从孕妇收集 胎龄），并且这些cTB/TB的全基因组谱分析可用于诊断胎儿遗传病。 异常在五年的资助期内，拟议的研究将通过两个具体的 目的：i）开发，优化和验证拟议的基于cTB的NIPD技术，ii）进行初始 在从UCLA和CSMC招募的孕妇中进行临床验证。联合小组设想， 所提出的基于cTB的NIPD技术的成功演示将引入一种革命性的NIPD 具有金标准诊断测试的灵敏度和特异性，而没有相关风险， 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.