Developmental regulation of HBV biosynthesis by Ten-eleven translocation (Tet) methylcytosine dioxygenases

十十一易位 (Tet) 甲基胞嘧啶双加氧酶对 HBV 生物合成的发育调节

• 批准号: 10733902
• 负责人: Alan McLachlan
• 金额: $ 39.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-09 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733902
• 关键词: Acute; Adult; Anabolism; Antiviral Therapy; Biological Models; Capsid; Cell Nucleus; Cell Therapy; Cessation of life; Chromatin Structure; Chromosome Deletion; Chronic; Chronic Hepatitis B; Circular DNA; Clinical; Cytoplasm; DNA; DNA Methylation; DNA sequencing; Development; Dioxygenases; Epigenetic Process; Gene Silencing; Genetic; Genetic Transcription; Genome; Genomic DNA; Genomics; Grant; Health; Hepatitis B Virus; Histones; Individual; Life Cycle Stages; Liver; LoxP-flanked allele; Mediating; Methylation; Molecular; Molecular Weight; Mus; Nuclear; Nucleotides; Physiological; Physiology; Predisposition; Protein translocation; Proteins; RNA; Regulation; Resolution; Reverse Transcription; Role; Signal Transduction; Suggestion; System; Tamoxifen; Transgenes; Transgenic Mice; Viral; Viral Genome; Viral Load result; Virus Diseases; Virus Inactivation; Virus Integration; Virus Replication; bisulfite; cellular targeting; chronic liver disease; demethylation; design; effective therapy; human pathogen; in vivo; inhibitor therapy; liver development; liver function; mouse model; new therapeutic target; novel; novel strategies; postnatal; prevent; recombinase; targeted treatment; therapeutic target

Project Summary and Relevance. Hepatitis B virus (HBV) infection is a worldwide health problem. It is estimated that there are 200 to 500 million HBV chronic carriers in the world for whom, to date, there is no reliable treatment. HBV causes both acute and chronic liver disease and is responsible for an estimated one million deaths annually. Currently available therapies reduce viral loads but fail to resolve chronic HBV infections. Therefore, effective treatments for chronic HBV infection are urgently required. The major obstacle to the resolution of chronic HBV infections is the eradication or inactivation of nuclear HBV covalently closed circular (ccc) DNA which is the template for viral transcription. To this end, we have developed HNF1α-null HBV transgenic mice and liver-specific Tet-deficient HBV transgenic mice. HNF1α-null HBV transgenic mice synthesize nuclear HBV cccDNA, a fraction of which is extensively methylated in adult mice. Liver-specific Tet-deficient HBV transgenic mice are viable, essentially display normal liver physiology, but lack detectable HBV transcription and replication (i.e. they are effectively “cured”) suggesting Tet is also essential for viral biosynthesis. The observation that Tet-deficient HBV transgenic mice fail to support HBV biosynthesis is consistent with the suggestion that Tet is essential for the developmental demethylation of HBV genomic DNA which epigenetically governs HBV transcription by modulating viral chromatin structure in vivo. Defining the precise temporal requirements for Tet expression associated with HBV transcription and replication will indicate the liver developmental stages when viral biosynthesis is susceptible to inhibition by Tet deficiency. This will be achieved by modulating Tet expression using our recently developed tamoxifen-inducible Tet-deficient HBV transgenic mouse model system. Using this system, the developmental control of HBV transcription, viral biosynthesis and HBV DNA methylation by Tet expression will be established and correlated with the epigenetic histone marks and chromatin structure associated with the HBV genome. Similar studies will be performed using the HNF1α-null Tet-deficient HBV transgenic mouse model of chronic viral infection so the developmental control of HBV transcription, viral biosynthesis, HBV DNA methylation, epigenetic histone marks and chromatin structure by Tet expression associated with the HBV genome can be compared between the HBV transgene DNA and the nuclear HBV cccDNA. Finally, the development of HBV transgenic mice supporting viral biosynthesis exclusively from extrachromosomal genomic DNA, a more physiologically relevant mouse model of chronic viral infection, will be developed so the effect of Tet-deficiency on HBV biosynthesis derived solely from HBV cccDNA can be determined. Defining the molecular signals responsible for the loss of HBV biosynthesis due to Tet deficiency may lead to the identification of cellular therapeutic targets, including but not restricted to Tet proteins, that are amenable to the development of novel small molecular weight therapeutic inhibitors to resolve rather than simply treat chronic HBV infection.

项目摘要和相关性。 丙型肝炎病毒（HBV）感染是全球健康问题。据估计有200至5亿 迄今为止，世界上HBV慢性载体没有可靠的待遇。 HBV引起急性和 慢性肝病，每年造成大约100万人死亡。目前可用 疗法减少病毒载荷，但无法解决慢性HBV感染。因此，有效的慢性治疗方法 紧急需要HBV感染。解决慢性HBV感染的主要障碍是 消除或灭活核HBV共价闭合（CCC）DNA，这是病毒的模板 转录。为此，我们开发了HNF1α-NULL HBV转基因小鼠和肝特异性TET缺陷型 HBV转基因小鼠。 HNF1α-NULL HBV转基因小鼠合成核HBV CCCDNA，其中一部分为 在成年小鼠中广泛甲基化。肝特异性TET缺陷HBV转基因小鼠可行，本质上是 显示正常的肝生理学，但缺乏可检测到的HBV转录和复制（即它们有效 “治愈”）表明TET对于病毒生物合成也是必不可少的。 TET缺陷HBV转基因的观察 小鼠无法支持HBV生物合成与TET对于发育至关重要的建议是一致的 HBV基因组DNA的脱甲基化，表观遗传通过调节病毒来控制HBV转录 体内染色质结构。定义与HBV相关的TET表达的精确临时要求 当病毒生物合成易受影响时，转录和复制将表明肝发育阶段 TET缺乏症的抑制作用。这将通过使用我们最近开发的TET表达来实现 他莫昔芬诱导的TET缺陷HBV转基因小鼠模型系统。使用此系统，发展 将通过TET表达控制HBV转录，病毒生物合成和HBV DNA甲基化 并与与HBV基因组相关的表观遗传组蛋白标记和染色质结构相关。 将使用HNF1α-NULL TET缺乏的HBV HBV转基因小鼠模型进行类似的研究 病毒感染，因此HBV转录，病毒生物合成，HBV DNA甲基化的发育控制， 通过与HBV基因组相关的TET表达的表观遗传组蛋白标记和染色质结构可以是 在HBV转化DNA和核HBV CCCDNA之间进行了比较。最后，HBV的发展 独家从外神体基因组DNA中支持病毒生物合成的转基因小鼠，更多 将开发出与生理相关的慢性病毒感染的小鼠模型，因此TET缺乏效率的影响 仅根据HBV CCCDNA得出的HBV生物合成。定义分子信号 负责因TET缺乏而导致的HBV生物合成丧失可能导致细胞的鉴定 治疗靶标，包括但不限于TET蛋白，这些蛋白适合新型的发展 小分子量疗法抑制剂可以解决，而不是简单地治疗慢性HBV感染。