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

• 批准号: 10675730
• 负责人: Osman Berk USTA
• 金额: $ 21万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/10675730
• 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; Liver; Marketing; Measurement; Measures; Messenger RNA; Methods; MicroRNAs; Modeling; Organ; Organoids; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Play; Population; Production; Rattus; Reporter; Research Personnel; Role; Route; Signal Transduction; Signaling Molecule; Surface; Technology; Testing; Three-dimensional analysis; Time; Tissues; Toxic Environmental Substances; Toxic effect; Toxicity Tests; Urea; Vesicle; Withdrawal; cohort; comparison control; cost; cytotoxicity; drug classification; early screening; exosome; experimental study; extracellular vesicles; improved; in vitro Assay; in vitro Model; in vivo; liver injury; medication safety; meter; nano-string; novel; organ on a chip; response; technology development; tissue culture

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.

肝毒性导致昂贵的晚期药物失败，其中25-40％的药物通过 第一阶段至第三阶段临床研究。尽管尽力确保药物安全，但仍然有相当数量的 毒品从市场上删除；主要原因是肝毒性，占所有肝毒性的约20-30％ 在过去的30年中，美国和欧盟的提款。研究人员一直在开发体外测定 和有助于预测人类状况响应毒品侮辱的标记，这是一种成功的桥梁方法 体外模型与体内条件之间的差距是难以捉摸的。因此，重要的是 技术和科学差距 - 就任何对任何人的体外反应而言 由于不同的端点测量值，药物可以预测其不良反应 - 仍然存在。在这种情况下， 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 独特的机会和一种统一的方法，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的潜在标记 在药物诱导的损伤和疾病下的细胞和组织的反应。因此，我们的长期目标是 开发整合的组织文化和外泌体分析平台，以便我们确定体外外泌体 与同一侮辱的体内签名相关的签名。我们的目标是开发 A）新型外泌体隔离和分类（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，以研究分类良好的药物的侮辱下研究外泌体特征， 并确定不良反应的最突出的签名。我们的中心假设是（a）外泌体 miRNA和mRNA是在药物损伤下检测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的细胞毒性（即潜毒，<ic50）和很多 比在体外的传统标记更早的时间点。这些是基于材料包装的观察 在外泌体中不是随机的，信号传导遗传材料（例如miRNA）在外泌体中更集中 与细胞细胞质相比。我们的理由是，通过外泌体检测细胞毒性打开 从长远来看，可以在体外和体内找到统一的特征。此外，早期的可能性 低剂量检测是制药公司的重要诱惑，其早期筛查通常 涉及毒性测试的短（几天）窗口，而不是慢性剂量研究。我们旨在测试我们的 通过两个综合目的的假设，首先开发一个框架来提取和分析肝的外泌体 球体，然后在慢性和急性药物挑战下调查外泌体特征。在 这项研究，我们希望表明外泌体货物可能是识别有毒侮辱的有效途径 低剂量和早期。然后可以将类似的方法用于环境毒素和其他侮辱。因此， 此处提出的研究的更广泛和长期影响直接影响人口的健康。