Mechanisms of HBV cccDNA transcriptional regulation in persons with and without HIV

HIV感染者和未感染者的HBV cccDNA转录调控机制

基本信息

批准号：
10882261
负责人：
ASHWIN BALAGOPAL
金额：
$ 81.1万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-14 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10882261
关键词：
Anti-Retroviral Agents Binding Biological Assay CD4 Positive T Lymphocytes Candidate Disease Gene Cause of Death Cell Count Cells Cessation of life Characteristics Chronic Chronic Hepatitis B Circular DNA Clinical Complementary DNA CpG Islands CpG dinucleotide Cytoplasm DNA Methylation Disease Outcome Epigenetic Process Funding Gene Expression Profiling Gene Silencing Genes Genetic Genetic Transcription Goals HIV HepG2 Hepatitis Hepatitis B Therapy Hepatitis B Virus Hepatocyte Human Immune Immune response In Vitro Individual Integration Host Factors Interruption Knowledge Liver Liver diseases Measurable Measures Messenger RNA Methods Modeling Modification Mutation Nonsense Codon Pathway interactions Persons Plasma Primary carcinoma of the liver cells Process RNA RNA Splicing Resolution Reverse Transcription Reverse Transcription Inhibition Sampling T-Cell Depletion Techniques Testing Therapeutic Tissues Transcript Transcriptional Regulation Use of New Techniques Variant Viral Viral Genes Viral hepatitis Virion Virus Diseases Virus Replication analog analytical method cDNA Library circular RNA co-infection digital end stage liver disease immune activation in vitro testing indexing insertion/deletion mutation interest intrahepatic laser capture microdissection multidisciplinary nanopore next generation sequencing novel response therapy development tool transcriptome transcriptome sequencing viral DNA viral RNA viral rebound virus genetics

项目摘要

Hepatitis B virus (HBV) infects >300 million people chronically and is the leading cause of end-stage liver disease and hepatocellular carcinoma (HCC), resulting in ~1 million deaths annually. HIV is a common co- infection in people with chronic hepatitis B (CHB) and worsens HBV and liver disease outcomes. Liver disease is a leading cause of death among PWH in the era of antiretrovirals, mostly due to viral hepatitis. Antiviral treatment with nucleos(t)ide analogues (NUCs) suppresses plasma HBV DNA but does not eliminate the covalently closed circular DNA (cccDNA) HBV template that resides in every infected cell. NUC interruption leads to HBV DNA rebound and clinical hepatitis. Therefore, finding an HBV cure is of major importance for HBV mono- and HIV/HBV co-infection. In the first funding cycle, we applied single-cell methods to HBV/HIV co-infected liver tissues and demonstrated that cccDNA is transcriptionally suppressed during NUCs. We now propose to understand the mechanism underlying this finding since permanently exploiting this mechanism therapeutically can lead to a functional cure. We propose an intensive study of NUC-associated cccDNA transcriptional suppression comparing people with HBV/HIV co- and HBV mono-infection. In aim one, we will quantify intrahepatic viral DNA and RNA quantities in hepatocytes from HBV mono- and HBV/HIV co-infected individuals using single-cell laser capture microdissection and our novel droplet digital PCR assays. We will quantify and compare the cccDNA transcriptional index (cccDNA TI), a measure of cccDNA transcription, in HBV mono- and HBV/HIV co- infection. In aim two we will use novel techniques to test whether cccDNA transcriptional suppression is due to genetic or epigenetic modifications of HBV using HBV mono- and HBV/HIV co-infected liver tissues from aim one. We will use Cas9-Nanopore sequencing to examine epigenetic modifications of CpG dinucleotides in cccDNA. We will use the NovaSeq platform to perform next-generation sequencing of cccDNA to look for mutations that may be associated with reduced transcription and to quantify global cccDNA transcriptional suppression of canonical viral genes and splice variants. In aim three we will test the hypothesis that the host response contributes to cccDNA transcriptional suppression and compare this response between HBV mono- and HBV/HIV co-infection. We will apply the novel 10X Visium spatial gene expression analysis (10x VSGEA) platform to liver tissue from Aim one to compare hepatocyte host transcriptomes in tissues with high vs. low cccDNA TI, directly interrogating HBV transcription using the same platform. We will confirm genes identified with 10X VSGEA using Tecan RNA sequencing, enriching for cells with high vs. low cccDNA TI. We will conduct in vitro studies to prove that genes of interest are associated with cccDNA transcriptional suppression, focusing on innate immune genes and HIV-associated immune activation. Our proposal fills knowledge gaps in understanding how to transform reversible cccDNA transcriptional suppression to a functional cure.

乙型肝炎病毒 (HBV) 慢性感染超过 3 亿人，是导致终末期肝病的主要原因疾病和肝细胞癌 (HCC)，每年导致约 100 万人死亡。 HIV 是一种常见的共慢性乙型肝炎 (CHB) 患者的感染会使乙型肝炎和肝病结果恶化。肝脏疾病在抗逆转录病毒药物时代，这是感染者死亡的主要原因，主要是由于病毒性肝炎。抗病毒物质核苷（酸）类似物（NUC）治疗可抑制血浆 HBV DNA，但不能消除存在于每个受感染细胞中的共价闭合环状 DNA (cccDNA) HBV 模板。 NUC中断导致 HBV DNA 反弹和临床肝炎。因此，寻找乙型肝炎治愈方法对于治疗乙型肝炎至关重要。 HBV 单一感染和 HIV/HBV 混合感染。在第一个资助周期中，我们将单细胞方法应用于 HBV/HIV 共感染的肝组织，并证明 cccDNA 在 NUC 期间转录受到抑制。我们现在建议了解该机制这一发现的基础是，因为在治疗上永久利用这种机制可以导致功能性治愈。我们建议对 NUC 相关 cccDNA 转录抑制进行深入研究，比较 HBV/HIV 双重感染和 HBV 单一感染者。在目标一中，我们将量化肝内病毒 DNA 并使用单细胞激光测定 HBV 单感染者和 HBV/HIV 共感染者肝细胞中的 RNA 数量捕获显微切割和我们新颖的液滴数字 PCR 检测。我们将对cccDNA进行量化和比较转录指数（cccDNA TI），在 HBV 单核和 HBV/HIV 共核中 cccDNA 转录的测量感染。在目标二中，我们将使用新技术来测试 cccDNA 转录抑制是否是由于使用来自 Target 的 HBV 单感染和 HBV/HIV 共感染肝组织对 HBV 进行遗传或表观遗传修饰一。我们将使用 Cas9-Nanopore 测序来检查 CpG 二核苷酸的表观遗传修饰 cccDNA。我们将使用NovaSeq平台对cccDNA进行新一代测序来寻找可能与转录减少相关的突变并量化全局 cccDNA 转录抑制典型病毒基因和剪接变体。在目标三中，我们将测试主机的假设反应有助于 cccDNA 转录抑制，并比较 HBV mono- 以及 HBV/HIV 双重感染。我们将应用新型 10X Visium 空间基因表达分析 (10x VSGEA) 从目标一到肝组织的平台，比较高与低组织中的肝细胞宿主转录组 cccDNA TI，使用同一平台直接询问 HBV 转录。我们将确认已识别的基因使用 Tecan RNA 测序的 10X VSGEA，富集具有高和低 cccDNA TI 的细胞。我们将进行体外研究以证明感兴趣的基因与 cccDNA 转录抑制相关，专注于先天免疫基因和艾滋病毒相关的免疫激活。我们的建议填补了以下方面的知识空白了解如何将可逆 cccDNA 转录抑制转化为功能性治愈。