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。慢性感染中与适应性免疫反应失败相关的一些特征已被确定，但在免疫反应失败之前和决定免疫反应失败的事件尚不清楚，可能涉及先天免疫反应。由于缺乏小动物模型，对丙肝病毒的研究一直受到阻碍。在本提案中，我们将利用GBV-B感染的狨猴，以一种创新的方法来评估参与先天免疫反应的一系列靶基因的敲除。GBV-B是与HCV关系最密切的病毒，可在狨猴和绢毛猴中诱发肝炎。该项目的中心假设是，先天免疫反应对于限制病毒在肝脏中的传播和协调适应性免疫反应以成功清除感染细胞至关重要。先天反应关键成分的敲低可能导致病毒血症持续时间延长和潜在的持续性感染。在与Santaris Pharma的合作中，我们最近在灵长类动物模型中展示了系统给药LNA（锁定核酸）寡核苷酸敲低基因的能力；黑猩猩体内microRNA-122的敲低有效地降低了HCV病毒载量。对于大多数研究项目来说，黑猩猩太大太贵了。狨猴是这个项目的理想选择，因为它们体型小，而且它们通常能清除GBV-B感染，这是与丙型肝炎病毒的少数区别之一。已经观察到超过一年的持续感染，因此持续能力显然是存在的。在目标1中，我们将使用与Santaris药物SPC3649相同的LNA反义寡核苷酸靶向miR122。这将作为一个积极的控制，以评估动力学和LNA敲低幅度在狨猴。在aim 2中，我们将敲低IL28B及其受体IL28RA。在男性中，IL28B的多态性可以高度预测HCV的持续感染，但我们对IL28B如何影响HCV感染知之甚少。在目标3中，我们将敲除TLR7， TLR7是浆细胞样树突状细胞上的主要受体，可感知病毒RNA并触发IFN的产生。我们还将敲低BST2，该受体向pDCs提供负反馈并控制IFN的产生。在目标4中，我们将敲除肝脏中导致IFN产生的病毒RNA传感器，包括RIG-I， TLR3和IRF3。尽管丙型肝炎病毒蛋白酶破坏了这两种途径，但它在体内病毒传播和肝脏isg诱导中的作用尚不清楚。总的来说，这些研究将有助于确定先天免疫反应的各个组成部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，持续的GBV-B感染可能是HCV疾病进展的一种新的灵长类模型。此外，开发一种靶向基因敲除的灵长类动物模型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.