Early Toxicity Detection Technologies via Exosomal Signatures in 3D Hepatic Tissues

通过 3D 肝组织中的外泌体特征进行早期毒性检测技术

基本信息

批准号：
10450330
负责人：
Osman Berk USTA
金额：
$ 25.2万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-02 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10450330
关键词：
3-Dimensional Accounting Acetaminophen Acute Adverse effects Adverse reactions Affect Albumins Animals Benchmarking Cells Cellular Assay Chronic Classification Clinical Research Coculture Techniques Control Groups Cytoplasm Detection Development Diagnostic Disease Dose Drug Interactions Drug Screening Drug Targeting Drug toxicity Ensure Enzymes Excision Failure Genetic Materials Goals Health Hepatic Hepatic Tissue Hepatocyte Hepatotoxicity Hour Human In Vitro Inflammatory Inflammatory Response Injury Kupffer Cells Light Liver Measurement Measures Messenger RNA Methods MicroRNAs Modeling Organ Organoids Pharmaceutical Preparations Pharmacologic Substance Phase Play Population Production Rattus Reporter Research Personnel Role Route Savings Signal Transduction Signaling Molecule Surface Technology Testing Three-dimensional analysis Time Tissues Toxic Environmental Substances Toxic effect Toxicity Tests Urea Vesicle Withdrawal base cohort cost cytotoxicity early screening exosome experimental study extracellular vesicles improved in vitro Assay in vitro Model in vivo liver injury medication safety nano-string novel organ on a chip response technology development tissue culture

项目摘要

Liver toxicity results in costly, late stage drug failures with 25-40% of drugs causing hepatic injuries through Phase I to Phase III clinical studies. Despite best efforts to ensure drug safety, there are still sizeable number of drug removals from the market; the primary reason being hepatotoxicity, which accounts for ~20-30% of all withdrawals in the US and EU over the last 30 years. While researchers have been developing in vitro assays and markers that help predict the human condition in response to drug insults, a successful approach to bridge the gap between the in vitro models and the in vivo condition has been elusive. Hence, the significant technological and scientific gap - in terms of correlating the in vitro responses to the in vivo conditions for any given drug to predict its adverse effects- still persists due to different end point measurements. In this context, exosomes - tiny (30-150 nm) vesicles that package genetic material and other signaling molecules - offer a unique opportunity and a unified approach in that they can be reliably measured both in in vitro and in vivo experiments. Moreover, recent studies strongly indicate that exosomes can be potential markers for adverse reactions of cells and tissues both under drug induced injuries and diseases. Accordingly, our long-term goal is to develop integrated tissue-culture and exosomal analysis platforms such that we identify in vitro exosomal signatures that are well correlated to in vivo signatures for the same insults. Our objective is to develop a framework of a) novel exosome isolation and classification (simultaneous size and surface markers) platform and b) stable 3D hepatic spheroid cultures to study exosomal signatures under insults from well classified drugs, and to identify the most prominent signatures for adverse reactions. Our central hypotheses are that (a) exosomal miRNAs, and mRNAs are potential markers for detection of cytotoxicity under drug insults, and furthermore (b) such exosomal signatures can be used to detect cytotoxicity at both low doses (i.e., subtoxic, <IC50) and much earlier time points than traditional markers in vitro. These are based on the observation that packing of materials in exosomes is not random and signaling genetic materials such as miRNAs are more concentrated in exosomes compared to the cellular cytoplasm. Our rationale is that detecting cytotoxicity with exosomes open up the possibility to find unifying signatures in vitro and in vivo in the long run. Furthermore, the possibility of early and low dose detection is a significant allure for pharmaceutical companies, whose early screening methods usually involve a short (few days) window of testing for toxicity rather than chronic dosing studies. We aim to test our hypotheses via two integrated aims, first, developing a framework to extract and analyze exosomes from hepatic spheroids, and, then, investigating exosomal signatures under chronic and acute drug challenges. At the end of this study, we expect to show that exosomal cargo can be a potent route to identify toxic insults and potentially at low doses and early on. Similar approaches can then be used for environmental toxins and other insults. Thus, the broader and long-term impact of the studies proposed here directly affect health of the human population.

肝毒性会导致代价高昂的晚期药物失败，25-40% 的药物会通过以下途径引起肝损伤： I期至III期临床研究。尽管尽了最大努力确保药品安全，但仍有相当数量的药品药品从市场上撤出；主要原因是肝毒性，约占所有毒性的 20-30% 过去 30 年美国和欧盟的提款情况。虽然研究人员一直在开发体外测定以及有助于预测人体对药物损伤的反应的标记物，这是一种成功的桥梁方法体外模型和体内条件之间的差距一直难以捉摸。因此，显着技术和科学差距 - 将体外反应与体内条件相关联给予药物来预测其不良反应 - 由于终点测量不同，仍然存在。在此背景下，外泌体 - 包装遗传物质和其他信号分子的微小（30-150 nm）囊泡 - 提供独特的机会和统一的方法，可以在体外和体内可靠地测量它们实验。此外，最近的研究强烈表明，外泌体可能是不良反应的潜在标志物。细胞和组织在药物引起的损伤和疾病下的反应。因此，我们的长期目标是开发集成的组织培养和外泌体分析平台，以便我们识别体外外泌体与相同侮辱的体内特征密切相关的特征。我们的目标是开发一个 a) 新型外泌体分离和分类（同时大小和表面标记）平台的框架 b) 稳定的 3D 肝球体培养物，用于研究分类良好的药物损伤下的外泌体特征，并确定不良反应最显着的特征。我们的中心假设是（a）外泌体 miRNA 和 mRNA 是检测药物损伤下细胞毒性的潜在标记，此外 (b) 这种外泌体特征可用于检测低剂量（即亚毒性，<IC50）和大量剂量下的细胞毒性。体外时间点比传统标记更早。这些是基于对材料包装的观察外泌体中的分布不是随机的，miRNA等信号遗传物质更集中在外泌体中与细胞质相比。我们的理由是，检测外泌体的细胞毒性可以打开从长远来看，有可能在体外和体内找到统一的特征。此外，早期和低剂量检测对制药公司来说具有巨大的吸引力，其早期筛选方法通常涉及短期（几天）的毒性测试而不是长期剂量研究。我们的目的是测试我们的通过两个综合目标提出假设，首先，开发一个从肝脏中提取和分析外泌体的框架球体，然后研究慢性和急性药物挑战下的外泌体特征。结束时在这项研究中，我们希望表明外泌体货物可以成为识别毒性损伤和潜在的潜在途径低剂量和早期。类似的方法可以用于环境毒素和其他损害。因此，这里提出的研究的更广泛和长期的影响直接影响到人类的健康。