Generation of a system enabling the study of hepatitis C entry factors in vivo and characterisation of HCV entry factor claudin-1

丙型肝炎病毒(丙型肝炎病毒)是全球范围内导致肝硬变和肝癌的主要原因。标准的治疗方案由聚乙二醇化的干扰素2a/2b和利巴韦林组成，根据不同的基因类型，蛋白酶抑制剂telapvir或bocpreviv。尽管已经取得了所有进展，但丙型肝炎病毒的治疗副作用丰富，并不能导致相当数量的患者产生持续的病毒学应答。开发新的治疗药物的基本前提是了解病毒的生命周期。病毒进入肝细胞是关键的一步。到目前为止，参与丙型肝炎病毒细胞进入的7个(共)受体(CD81、SCARB1、OLCN、NPC1L1、EGRF、EphA2和CLDN1)已被确定。本项目旨在通过建立一个基因人源化的小鼠模型来进一步阐明CLDN1在丙型肝炎病毒细胞进入中的作用。这个模型是基于先前的观察，即自然对丙型肝炎病毒不敏感的小鼠，可以通过腺病毒表达人CD81和OCLN来使细胞培养产生丙型肝炎病毒(HCVcc-FlpE)。HCVcc-FlpE的基因组编码FlpE-重组酶，并在rosa26-FSF-F2AY报告小鼠中激活黄色荧光蛋白报告基因，作为感染的读出。这个以改良形式建立的人源化小鼠模型现在将与条件基因消融技术相结合。具有CLDN1基因外显子的小鼠的繁殖目前正在进行中。这些小鼠将与现有的在白蛋白启动子下表达Cre重组酶的缺失菌株杂交，并导致肝脏特异的CLDN1基因敲除。这一步骤是必要的，因为完全纯合CLDN1缺乏症会导致小鼠新生儿致死表型。首先，我们的目标是确定肝脏CLDN1缺乏症的特征。我们将特别解决这个问题，即claudin家族其他成员的表达谱是如何改变的，以及他们是否可以采用CLDN1的生理作用。下一步将是通过肝脏特异性CLDN1的敲除来跨越报告鼠线。由此产生的后代将与人CD81和OCLN以及人或小鼠CLDN1进行转导。在注射HCVcc-FlpE后，我们将定量报道者的活性。这种方法将允许检查每个给定基因对丙型肝炎病毒感染的影响，即使相应的敲除会导致致命的表型。

Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. The standard therapeutic regime consists of pegylated interferon 2a/2b and ribavirin and depending on the genotype the protease inhibitors telaprevir or boceprevir.Despite all progress that has been done, treatment of HCV is rich of side effects and does not lead to a sustained virological response for a significant number of patients.An elemental prerequisite for the development of new therapeutic agents is the understanding of the viral life cycle. A crucial step is the virus entry into the hepatocyte.Until now seven (co-)receptors, that are involved in HCV cell entry (CD81, SCARB1, OLCN, NPC1L1, EGRF, EphA2 and CLDN1), have been identified.The proposed project aims to further elucidate the role of CLDN1 in HCV cell entry by using a genetically humanized mouse model. This model is based on the previous observation, that mice, which are naturally not susceptible for HCV, can be rendered permissive to cell culture derived HCV (HCVcc-FlpE) by adenoviral expression of human CD 81 and OCLN. The genome of HCVcc-FlpE encodes for a FlpE-recombinase and activates a yellow fluorescent protein reporter in Rosa26-FSF-F2AY reporter mice, which is used as a read-out for infection.This genetically humanized mouse model, which is well established in a modified form will now be combined with the technique of conditional gene ablation. The gerneration of mice with floxed exons in the CLDN1 locus is currently under progress. These mice will be crossed to an already existing deleter strain expressing CRE recombinase under an albumin promoter and leading to a liver-specific knockout of the CLDN1 gene. This step is necessary as a complete homozygous CLDN1 deficiency results in a neonatally lethal phenotype in mice.In the first instance we aim to characterize the hepatic CLDN1 deficiency. We will particularly address the question, how the expression profile of other members of the claudin family is altered and whether they can adopt the physiological role of CLDN1.The next step will be to cross the reporter mouse line with the liver-specific knockout of CLDN1. The resulting progeny will be transduced with human CD81 and OCLN and also with human or murine CLDN1. After injection of HCVcc-FlpE we will quantify reporter activity.This approach will allow to examine the impact of every given gene on HCV infection, even if the corresponding knockout would lead to a lethal phenotype.