Non-Invasive Prenatal Diagnostics Based on Circulating Trophoblasts

基于循环滋养细胞的无创产前诊断

• 批准号: 10675005
• 负责人: Margareta Pisarska
• 金额: $ 79.3万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10675005
• 关键词: Acceleration; Adopted; Advanced Development; Amniocentesis; Aneuploidy; Biological Markers; Biological Models; Biometry; Blood; Blood Cells; Blood specimen; Cell Separation; Cells; Cervix Uteri; Chemistry; Chorionic Villi Sampling; Chromosome abnormality; Clinic; Clinical; Collaborations; Collecting Cell; Collection; Computer software; DNA; DNA amplification; Data; Detection; Diagnosis; Diagnostic; Diagnostic tests; Discipline of obstetrics; Down Syndrome; Exclusion; Exhibits; Fetus; First Pregnancy Trimester; Funding; Genetic Diseases; Genome; Genomic DNA; Genotype; Gestational Age; Goals; Harvest; In Vitro; Individual; Interdisciplinary Study; Joints; Karyotype; Karyotype determination procedure; Longevity; Medical center; Methods; Microarray Analysis; Microfluidics; Molecular Abnormality; Monitor; Motivation; NanoVelcro; Nanostructures; Neoplasm Circulating Cells; Pathology; Performance; Pregnancy; Pregnant Women; Prenatal Diagnosis; Procedures; Process; Public Health; Reproducibility; Research; Resolution; Risk; Sampling; Sensitivity and Specificity; Spontaneous abortion; Surface; Swab; Testing; Validation; biomarker signature; cell free fetal DNA; clinical application; cohort; cost efficient; diagnostic platform; diagnostic strategy; diagnostic technologies; diagnostic value; fabrication; feasibility testing; fetal; fetal diagnosis; fetus cell; genetic disorder diagnosis; genetic testing; genome-wide; immunocytochemistry; imprint; improved; innovation; laser capture microdissection; microdeletion; microscopic imaging; nanoimprinting; nanomaterials; next generation sequencing; prenatal; prenatal testing; preservation; recruit; screening; single cell analysis; specific biomarkers; success; trophoblast; whole genome

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.

项目摘要/摘要 该修订后的U01提案的长期目标是进行先进的开发和严格验证 基于新兴的循环滋养细胞（CTB）的非侵入性产前诊断（NIPD）技术，能够 i）从母体血液中富集/计数的CTB，ii）隔离单个CTB，用于全基因组检测 怀孕前三个月的胎儿遗传异常。替代研究计划也是 提出了探索相同工作流的使用来隔离和表征子宫颈中的滋养细胞（TBS） 样品。 在母体血液中潜在的循环胎儿核细胞（CFNC）中，CTB是理想的目标 考虑他们的（i）寿命短，不包括先前怀孕或 流产，（ii）胎儿核型和基因型的表示，以及（iii）独特集合的表达 可用于富集和识别的生物标志物。但是，隔离纯CTB已 由于它们的丰度极低，因此在技术上挑战。 在过去的十年中，Tseng博士在加州大学洛杉矶分校的研究团队开发 诊断平台（又名Nanovelcro芯片）。为了利用纳米诺维克罗芯片的NIPD实用程序，团队首先 开发了一种纳米印刷制造过程，以准备激光捕获微分解（LCM） - 兼容 纳米底物的成本效益和可扩展方式。这些芯片，结合使用捕获和 免疫细胞化学（ICC）剂，表现出出色的CTB捕获性能。并行高分辨率 已经开发了显微镜成像和分析软件，以识别和注册单个CTB 底物，可以通过LCM高度准确地分离单个CTB。与比萨斯卡博士合作 联合团队展示了从血液处理，单个CTB隔离和DNA扩增开始的工作流程， 一直通过ArrayCGH和/或下一代测序通过CTB的整个基因组分析。 我们的中心假设是，可以从5毫升的母体血液中收集> 10 ctbs（> 50 tbs 子宫颈样品），在怀孕的头三个月从孕妇那里收集（8-12周 胎龄），这些CTB/TBS的整个基因组分析可用于诊断胎儿遗传 异常。在5年的资金期间，拟议的研究将通过两个特定的 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 从UCLA和CSMC招募的孕妇的临床验证。联合团队设想 拟议的基于CTB的NIPD技术的成功演示将引入革命性的NIPD 具有黄金标准诊断测试的灵敏度和特异性的解决方案，而没有相关风险 胎儿。

项目成果

High-throughput mRNA-seq atlas of human placenta shows vast transcriptome remodeling from first to third trimester.

人类胎盘的高通量 mRNA-seq 图谱显示从妊娠早期到晚期的巨大转录组重塑。

• DOI: 10.1101/2023.06.06.543972
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Gonzalez,TaniaL;Wertheimer,Sahar;Flowers,AmyE;Wang,Yizhou;Santiskulvong,Chintda;Clark,EkaterinaL;Jefferies,CarolineA;Lawrenson,Kate;Chan,JessicaL;Joshi,NikhilV;Zhu,Yazhen;Tseng,Hsian-Rong;Karumanchi,SAnanth;Williams,John