Exosome platforms for assessment and therapy of chronic liver disease

 描述(由申请者提供)：广泛的长期目标是改进肝脏疾病评估和治疗的方法。大约有550万美国成年人(即美国成年人口的2%-3%)患有慢性肝病。酒精性肝病(ALD)在西方慢性肝病中所占比例最大，并加剧了非酒精性原因造成的肝脏损伤。酒精性肝硬变在美国最常见的死亡原因中排名第八，在胃肠道相关死亡中排名第二。有一种尚未得到满足的需求，即通过使用现代分子方法来分析可以以微创或非侵入性方式收集的生物标本，来改进我们的诊断选择，而新的领先优势已经随着“外显体”的发现而出现。这些是由许多类型的细胞分泌的纳米囊，其中含有反映其产生细胞的转录和/或翻译活动的microRNAs、mRNAs和蛋白质的复杂混合物。我们发现：(1)小鼠实验性肝纤维化过程中，外周血中miRs的动态变化；(Ii)肝星状细胞(HSC)分泌的纤维化相关miRs或mRNA的表达反映了细胞内纤维化途径的诱导状态；(Iii)调控exosome生物发生的分子在HC中的表达是酒精依赖的；(Iv)与纤维化相关的miRs在HSC之间穿梭，并被受体细胞摄取后发挥功能；(V)HSC的外切体在体内和体外都优先被HSC摄取，这突显了HSC来源的外切体存在一种新的归巢机制。因此，我们的总体目标是确定外切体在肝脏疾病中的作用及其在疾病中的潜在作用。 诊断或治疗。我们的中心假设是，外切体对肝病有诊断和治疗作用，包括酒精引起的肝病。检验我们的假设的目的是：具体目标1：建立乙醇介导的外切体生产和分子有效载荷的变化。我们将确定乙醇对HSC外切体生物发生和分泌途径的影响，并从ALD患者的血清中建立外切体的miR特征。2.建立外切体归巢到HSC的机制和治疗应用。我们将表征整合素介导的外切小体和HSC之间的相互作用，并在实验性肝纤维化的体内模型中，确定它们在介导HSC特异性定位和携带抗纤维化siCTGF的外切小体的治疗效果中的作用。这些探索性R21研究的预期结果将是通过改善对外切体功能的理解和开发，在慢性肝病的诊断和治疗方面取得创新突破。这项应用的基本原理是，识别胞外体分子有效载荷和细胞间穿梭机制将与疾病诊断和治疗直接相关。这些研究的积极影响是，它们将改善全球数百万患有ALD和其他肝脏疾病的人的健康。

 DESCRIPTION (provided by applicant): The broad long-term objective is to improve methods of disease assessment and treatment in the liver. Approximately 5.5 million American adults (i.e., 2-3% of the adult US population) suffer from chronic liver disease. Alcoholic liver disease (ALD) accounts for the largest proportion of chronic liver disease in the West and exacerbates liver injury due to non-alcoholic causes. Alcoholic cirrhosis ranks as the 8th most common cause of mortality in the US and the second leading cause of GI-related deaths. There is an unmet need to improve our arsenal of diagnostic options by using modern molecular approaches to analyze biological specimens that can be collected in a minimally-invasive or non-invasive manner, and a new lead has emerged with the discovery of "exosomes". These are nanovesicles that are secreted by many cell types and which contain a complex mixture of microRNAs, mRNAs and proteins that reflect the transcriptional and/or translational activity of their producer cells. We showed that (i) miRs in circulating exosomes undergo dynamic changes during experimental fibrosis in mice; (ii) expression of fibrosis-related miRs or mRNA in exosomes secreted by hepatic stellate cells (HSC, the principal pro-fibrogenic cell type in the liver) reflects the induction status of fibrogenic pathways within the cells; (iii) expression in HC of molecules regulating exosome biogenesis is ethanol-dependent; (iv) fibrosis-related miRs are exosomally shuttled between HSC and are functional after being taken up by recipient cells; and (v) exosomes from HSC are preferentially taken up by HSC both in vivo and in vitro, highlighting the existence of a novel homing mechanism for HSC-derived exosomes. Thus our overall objective is to establish the role of exosomes in liver disease and their potential role in disease diagnosis or therapy. Our central hypothesis is that exosomes have diagnostic and therapeutic utility in liver disease, including that caused by alcohol. The Aims to test our hypothesis are: Specific Aim 1: Establish ethanol-mediated changes in exosome production and molecular payload. We will determine the effect of ethanol on pathways of exosome biogenesis and secretion in HSC and establish miR signatures in exosomes from the serum of ALD patients Specific Aim 2. Establish mechanisms and therapeutic applications of exosome homing to HSC. We will characterize integrin-mediated interactions between exosomes and HSC and, in an in vivo model of experimental liver fibrosis, establish their role in mediating HSC-specific localization and therapeutic efficacy of exosomes loaded with anti-fibrotic siCTGF. The expected outcome of these exploratory R21 studies will be innovative breakthroughs in the diagnosis and therapy of chronic liver disease by improved understanding and exploitation of exosome function. The rationale underlying the application is that identification of exosomal molecular payloads and intercellular shuttling mechanisms will have direct relevance to disease diagnosis and therapy. The positive impact of these studies is that they will improve the health of millions of people world-wide with ALD and other liver diseases.