The Innate Immune Response in the Marmoset Model of GBV-B Infections: A Surrogate

描述（由申请人提供）：丙型肝炎病毒感染影响约2%的人口，通常进展为肝硬化和肝癌。与慢性感染中失败的适应性免疫应答相关的一些特征已经被确定，但是在失败的免疫应答之前和确定失败的免疫应答的事件不太清楚，并且可能涉及先天免疫应答。由于缺乏小动物模型，对HCV的研究一直受到阻碍。在这个建议中，我们将使用GBV-B感染的绒猴在一个创新的方法来评估敲除的一系列靶基因参与先天免疫反应。GBV-B是与丙型肝炎病毒关系最密切的病毒，它会诱导绒猴和绢毛猴发生肝炎。该项目的中心假设是先天免疫应答对于限制病毒在肝脏中的传播和协调适应性免疫应答以成功清除感染细胞是必不可少的。先天反应的关键组分的敲低可能导致病毒血症持续时间延长和潜在的持续感染。与Santaris Pharma合作，我们最近证明了全身给予LNA（锁核酸）寡核苷酸在灵长类动物模型中敲低基因的能力;黑猩猩中microRNA-122的敲低有效地降低了HCV病毒载量。黑猩猩太大，太昂贵，无法用于大多数研究项目。绒猴是这个项目的理想选择，因为它们体积小，而且它们通常可以清除GBV-B感染，这是与HCV的少数差异之一。已观察到持续感染超过一年，因此持续感染的能力显然存在。在目标1中，我们将使用与Santaris药物SPC 3649相同的LNA反义寡核苷酸靶向miR 122。这将用作阳性对照，以评价绒猴中LNA敲低的动力学和幅度。在目标2中，我们将敲低IL 28 B及其受体IL 28 RA。在人类中，IL 28 B的多态性高度预测HCV的持续感染，但我们对IL 28 B如何影响HCV感染知之甚少。在目标3中，我们将敲低TLR 7，TLR 7是浆细胞样树突状细胞上感知病毒RNA并触发IFN产生的主要受体。我们还将敲除BST 2，该受体为pDC提供负反馈并控制IFN产生。在目标4中，我们将敲低肝脏中的病毒RNA传感器，导致IFN产生，包括RIG-I、TLR 3和IRF 3。尽管HCV蛋白酶破坏了这两种途径，但其在体内病毒传播和肝脏中ISG诱导中的作用仍知之甚少。总的来说，这些研究将有助于确定先天免疫反应的各种成分在病毒清除中的作用，并可能为慢性感染的治疗提供新的方法。此外，持续性GBV-B感染将代表HCV疾病进展的新灵长类动物模型。此外，靶向基因敲除的灵长类动物模型的开发将在许多研究领域具有重要价值。

DESCRIPTION (provided by applicant): Hepatitis C virus infections affect approximately 2% of the population and often progress to cirrhosis and liver cancer. Some of the characteristics associated with a failed adaptive immune response in chronic infection have been identified, yet the events that precede and determine the failed immune response are less clear and may involve the innate immune response. Research on HCV has been impeded by the lack of a small animal model. In this proposal, we will use GBV-B infected marmosets in an innovative approach to evaluate knockdown of a series of target genes involved in the innate immune response. GBV-B is the virus most closely related to HCV and it induces hepatitis in marmosets and tamarins. The central hypothesis of this project is that the innate immune response is essential for limiting viral spread in the liver and for orchestrating the adaptive immune response for the successful clearance of infected cells. Knockdown of critical components of the innate response may lead to extended duration of viremia and potentially persistent infections. In collaboration with Santaris Pharma, we recently demonstrated the ability of systemically administered LNA (locked nucleic acid) oligos to knockdown genes in a primate model; the knockdown of microRNA-122 in chimpanzees effectively reduced HCV viral load. Chimpanzees are too large and expensive for use in most research projects. Marmosets are ideal for this project due to the small size and the fact that they normally clear GBV-B infection, one of the few differences from HCV. Persistent infections beyond one year have been observed, thus the capacity for persistence clearly exists. In aim 1, we will target miR122 using the same LNA antisense oligo as the Santaris drug, SPC3649. This will serve as a positive control to evaluate the kinetics and magnitude of LNA knockdown in the marmoset. In aim 2, we will knockdown IL28B and its receptor IL28RA. In man polymorphisms in IL28B are highly predictive of persistent infection with HCV, yet we have little understanding of how IL28B influences HCV infection. In aim 3, we will knockdown TLR7, the primary receptor on plasmacytoid dendritic cells sensing viral RNA and triggering IFN production. We will also knockdown BST2, the receptor that provides negative feedback to pDCs and controls IFN production. In aim 4, we will knockdown the sensors of viral RNA in the liver leading to IFN production including RIG-I, TLR3 and IRF3. Although, HCV protease subverts both these pathways, the role this plays in vivo in the spread of virus and induction of ISGs in the liver is poorly understood. Collectively, these studies will help define the role of various components of the innate immune response in viral clearance and potentially new approaches to therapies for chronic infections. In addition, persistent GBV-B infections would represent a new primate model for HCV disease progression. Furthermore, development of a primate model of targeted gene knockdown would be of great value in a number of research areas.