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

肝毒性导致昂贵的晚期药物失败，其中25-40%的药物通过以下途径引起肝损伤： I期至III期临床研究。尽管已尽最大努力确保药物安全，但仍有相当数量的 从市场上撤下药物;主要原因是肝毒性，占所有药物的约20-30% 在过去的30年里，美国和欧盟的退出。虽然研究人员一直在开发体外检测方法， 和标记物，帮助预测人类对药物侮辱的反应，这是一种成功的方法， 体外模型和体内条件之间的差距是难以捉摸的。因此， 技术和科学的差距-在相关的体外反应，在体内条件的任何 给予药物以预测其不良反应-由于不同的终点测量结果仍然存在。在这一背景下， 外泌体是一种微小的（30-150 nm）囊泡，包装遗传物质和其他信号分子， 独特的机会和统一的方法，因为它们可以在体外和体内可靠地测量 实验此外，最近的研究强烈表明，外泌体可以是不良反应的潜在标志物。 在药物引起的损伤和疾病下细胞和组织的反应。因此，我们的长期目标是 开发整合的组织培养和外泌体分析平台， 与相同损伤的体内特征良好相关的特征。我们的目标是发展一个 a）新的外泌体分离和分类（同时的大小和表面标志物）平台的框架 和B）稳定的3D肝球状体培养物以研究在良好分类的药物的损伤下的外来体特征， 并识别最显著的不良反应特征。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志物，此外（B） 这种外来体标记可用于检测两种低剂量下的细胞毒性（即，亚毒性，<IC 50）和 比传统的体外标记物更早的时间点。这些都是基于观察，包装材料 外泌体中的信号传导不是随机的，并且信号传导遗传物质如miRNA更集中于外泌体中 与细胞质相比。我们的基本原理是，检测外泌体的细胞毒性， 从长远来看，有可能在体外和体内找到统一的签名。此外，早期和 低剂量检测对制药公司来说是一个重要的诱惑，他们的早期筛查方法通常 涉及短期（几天）毒性试验窗口，而不是长期给药研究。我们的目标是测试我们的 假设通过两个综合目标，首先，开发一个框架，从肝细胞中提取和分析外泌体， 球状体，然后研究慢性和急性药物挑战下的外泌体签名。结束时 这项研究，我们希望表明，外泌体货物可以是一个有效的途径，以确定有毒的侮辱和潜在的 在低剂量和早期。类似的方法可以用于环境毒素和其他侮辱。因此，在本发明中， 这里提议的研究的广泛和长期影响直接影响到人类的健康。

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.