IL28B基因通过JAK-STAT通路激活干扰素刺激基因清除HBV cccDNA的作用机制研究

Infection of hepatitis B virus (HBV) is one of the most serious and prevalent global health problems. Its pathogenesis is complex, and the treatment is still the problem today. Covalently closed circular DNA (cccDNA) exists persistently exist in the liver is the cause of treatment failure and relapse of HBV. Our previous work has successfully constructed a recombinant cccDNA model in vitro and in vivo，and we found the levels of IL28B gene mRNA and cccDNA were negatively correlated, through the insoluble cellular pathways remove cccDNA, and result in antiviral treatment response. Further mechanism analysis suggests that higher expression level of IL28B phosphorylated STAT1 protein, activation of downstream interferon-stimulated genes (ISG). Therefore, on the base of our previous work, we proposed the scientific hypothesis that minicircle DNA-mediated liver-specific high expression of IL28B activated JAK-STAT signaling pathway, activation of downstream target genes ISG, and lead to clear cccDNA, which play an important role of anti-HBV. In order to verify this hypothesis. The project intends to construct IL28B liver-specific expression of minicircle DNA, investigate the biological behavior of its anti-HBV biological behaviour, and demonstrate it specific mechanism of clear cccDNA. Further validation in animal model to confirm that IL28B clear cccDNA result in anti-HBV, as well as exploring its potential role in HBV targeted therapy.

乙型肝炎病毒（HBV）感染严重威胁人类健康，是全球公共卫生问题。其发病机制复杂，治疗仍是当今的难题。共价闭合环状DNA(cccDNA)的存在及不彻底清除是HBV治疗失败及复发的根本原因。申报人前期工作中成功构建重组cccDNA模型，同时发现IL28B基因与病毒性肝炎抗病毒治疗应答密切相关。而IL28B基因作为干扰素新家族成员IFN-λ中的一员，可能通过机体内非溶解细胞途径抑制或清除cccDNA。因此，我们基于前期工作，提出“微环DNA介导肝脏特异性表达IL28B通过活化JAK-STAT信号通路，激活下游ISG靶基因，清除cccDNA，从而发挥抗HBV的作用”的科学假设，拟通过构建肝脏特异性表达IL28B的微环DNA，探讨其抗HBV生物学行为，并明确其清除cccDNA的具体作用机制。进一步在动物水平验证IL28B清除cccDNA而抗HBV的作用，探讨其潜在的靶向治疗作用。

乙型肝炎病毒（HBV）共价闭合环状DNA(cccDNA)的存在及不彻底清除是HBV治疗失败及复发的根本原因。本项目基于微环DNA技术构建重组乙肝病毒共价闭合环状DNA（HBV rcccDNA），通过一系列功能研究验证HBV rcccDNA在HuH7细胞的复制能力，并在树鼩中成功验证该HBV rcccDNA具有感染能力，最后通过Crispr/Cas9系统编辑技术证实可以有效的清除cccDNA。研究结果表明该重组cccDNA无论在结构还是功能上均跟野生型cccDNA非常接近：重组cccDNA能介导病毒基因组复制、病毒基因表达，并能产生完整的感染性病毒颗粒。该研究结果有望助力乙肝病毒cccDNA的生物学研究及抗cccDNA药物的研发。基于以上rcccDNA细胞动物模型，通过系列功能试验，获得MC-IL28B影响HbsAg、HBeAg表达及HBV DNA复制，阐明MC-IL28B通过磷酸化 STAT1、STAT2调节ISG基因的转录水平的分子机制。该研究结果为IL28B应用于抗HBV治疗提供理论依据和临床证据。