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

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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.