HCV biology and inhibition visualized at the single molecule level

HCV 生物学和抑制在单分子水平上可视化

• 批准号: 8641463
• 负责人: NILS G WALTER
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-15 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8641463
• 关键词: 5' Untranslated Regions; Adopted; Adverse effects; Adverse event; Affect; Antiviral Agents; Behavior; Binding; Biology; Cell Culture Techniques; Cells; Cessation of life; Chronic Hepatitis; Collaborations; Color; Cultured Cells; Cytoplasmic Granules; Dependence; Diffusion; Effectiveness; Ensure; Evolution; FDA approved; Family; Flaviviridae; Gene Expression Regulation; Genome; Goals; HIV; Hepatitis C virus; Hepatocyte; Infection; Interferons; Label; Life; Liver Cirrhosis; Mediating; Messenger RNA; MicroRNAs; Microinjections; Patients; Pattern; Performance; Pharmaceutical Preparations; Play; Primary carcinoma of the liver cells; Process; Protease Inhibitor; Proteins; RNA; RNA Interference; RNA Viruses; Reaction; Recruitment Activity; Resolution; Ribavirin; Risk; Role; Small Interfering RNA; Small RNA; Testing; Therapeutic; Time; Titrations; Universities; Ursidae Family; Vaccines; Viral; Virus Replication; War; anti-hepatitis C; chronic liver disease; cyanine dye 5; fluorophore; improved; innovation; knock-down; mRNA Transcript Degradation; novel; particle; protein complex; public health relevance; research study; single molecule; small hairpin RNA; spatiotemporal; standard care; standard of care; stoichiometry; trafficking; viral RNA

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus of the Flaviviridae family that causes more deaths in the US than HIV. Infection with HCV is the leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, and currently affects ~180 million people worldwide, ~4 million of whom reside in the US. No protective vaccines exist against HCV. The current standard of care is a combination of pegylated interferon and ribavirin, with limited effectiveness and severe side effects. The recently FDA- approved protease inhibitors Telaprevir and Boceprevir have to be administered with this standard treatment to achieve sustained viral clearance so that even the newest therapy is still associated with serious side effects. The recent discovery by our collaborator Peter Sarnow (Stanford University) of a remarkable protective role for microRNA (miRNA) miR-122 in HCV replication has paved the way for anti-miRs, which inhibit endogenous miRNAs, as a novel class of HCV drugs. In addition, our collaborator Brian Johnston from SomaGenics Inc. recently developed several small interfering RNAs (siRNAs) as well as short small hairpin RNAs (sshRNAs) that mediate efficient RNA interference (RNAi) against HCV in cell culture. The primary objective of this R21 proposal is to dissect the underlying intracellular mechanisms of these promising small-RNA anti-HCV drugs, with the long-term goal of further improving their efficacy. To this end, we will bring to bear the powerful capabilities of Single molecule, High-Resolution Localization and Counting (iSHiRLoC), an innovative probe concept we recently developed for detecting the trafficking and assembly of single miRNA molecules inside live cultured cells. iSHiRLoC entails single particle tracking of physiologically relevant lw numbers of microinjected, inconspicuously fluorophore-labeled RNAs to record their spatiotemporal distribution and count their stoichiometry upon RNA:protein (RNP) complex assembly. We propose to expand iSHiRLoC to two colors and study HCV biology and inhibition at an unprecedented single molecule level, by pursuing the following three Specific Aims: (i) We will probe the mechanism of miR-122-mediated intracellular protection of HCV RNA by using two-color iSHiRLoC in cultured, miR-122-free HepG2 hepatocytes and comparing the spatiotemporal distributions and assembly stoichiometries of Cy5-labeled miR-122, microinjected in conjunction with either Cy3-labeled subgenomic HCV RNA or Cy3-labeled AldoA target mRNA. (ii) We will probe the mechanism of siRNA- and sshRNA-mediated intracellular RNA interference against HCV by applying two-color iSHiRLoC to siRNAs and sshRNAs already validated against HCV in hepatocytes by SomaGenics Inc. (iii) We will observe the "tug-of-war" between miR-122-mediated protection of and RNAi against HCV RNA by performing a precise intracellular "titration" of Cy3-labeled HCV RNA with varying amounts of unlabeled miR-122 in the presence of our most effective Cy5-labeled siRNA/shRNA to directly observe the evolution of the HCV RNA spatiotemporal distribution as the "tug-of-war" between the two small RNAs plays out.

描述（由申请人提供）：丙型肝炎病毒（HCV）是黄病毒科的一种正义单链RNA病毒，在美国造成的死亡人数超过HIV。HCV感染是慢性肝炎、肝硬化和肝细胞癌的主要原因，目前影响全球约1.8亿人，其中约400万人居住在美国。没有针对HCV的保护性疫苗。目前的标准治疗是聚乙二醇干扰素和利巴韦林的组合，有效性有限且副作用严重。最近FDA批准的蛋白酶抑制剂Telaprevir和Boceprevir必须与这种标准治疗一起施用，以实现持续的病毒清除，使得即使是最新的治疗仍然与严重的副作用相关。我们的合作者Peter萨尔诺（斯坦福大学）最近发现microRNA（miRNA）miR-122在HCV复制中具有显著的保护作用，这为抑制内源性miRNA的抗miR作为一类新型HCV药物铺平了道路。此外，我们的合作者来自SomaGenics Inc.的Brian约翰斯顿。最近开发了几种小干扰RNA（siRNA）以及短的小发夹RNA（sshRNA），其在细胞培养物中介导针对HCV的有效RNA干扰（RNAi）。这项R21提案的主要目标是剖析潜在的细胞内 这些有前途的小RNA抗HCV药物的机制，长期目标是进一步提高其疗效。为此，我们将利用单分子高分辨率定位和计数（iSHiRLoC）的强大功能，这是我们最近开发的一种创新探针概念，用于检测活培养细胞内单个miRNA分子的运输和组装。iSHiRLoC需要对生理学相关的lw数量的显微注射的、不明显的荧光团标记的RNA进行单粒子跟踪，以记录它们的时空分布并计算它们在RNA：蛋白质（RNP）复合物组装时的化学计量。我们建议将iSHiRLoC扩展到两种颜色，并在前所未有的单分子水平上研究HCV生物学和抑制作用，具体目标如下：（i）我们将通过使用双色iSHiRLoC在培养的，无miR-122的HepG 2肝细胞，并比较Cy 5标记的miR-122的时空分布和组装化学计量，与Cy 3标记的亚基因组HCV RNA或Cy 3标记的AldoA靶mRNA一起显微注射。(ii)我们将通过将双色iSHiRLoC应用于已经由SomaGenics Inc.在肝细胞中验证的针对HCV的siRNA和sshRNA，来探索siRNA和sshRNA介导的针对HCV的细胞内RNA干扰的机制。(iii)我们将通过在我们最有效的Cy 5标记的siRNA/shRNA存在下，用不同量的未标记的miR-122对Cy 3标记的HCV RNA进行精确的细胞内“滴定”来观察miR-122介导的保护和RNAi对HCV RNA的“拔河”，以直接观察HCV RNA时空分布的演变，因为这两种小RNA之间的“拔河”发挥作用。