“点击↑↑↑，关注临床科研”
前言
根据世界卫生组织资料，2015年世界有2.57亿人患有慢性乙型肝炎（定义为乙型肝炎表面抗原阳性），约88.7万人由于乙肝死亡，主要死于肝硬化和肝癌。根据中国卫计委于2015年公布的信息，中国有9,000万乙肝病毒感染者。
沉重的疾病负担，迫使科学家全力寻找提高乙肝疗效、甚至治愈乙肝的办法。
为此，我们解读美国在乙肝研究中资助的基金情况，期望从大数据分析中，找到一丝临床科研发展的规律、或得到一些启发。https://www.who.int/news-room/fact-sheets/detail/hepatitis-bhttp://www.gov.cn/xinwen/2015-07/28/content_2904192.htm检索词：研究课题的题目中有"hepatitis B" "HBV" "Hep B"的基金；
分析机构：美国恒祥咨询公司（Healsan Consulting）。
检索结果：
从1985年开始的记录看，针对乙肝的资助项目从1985年的63项，一直到2020年达到71项。
累计资助2,059项HBV研究，累计资助基金超过5亿美元。
历年来资助的研究中，以T细胞、艾滋病、DNA聚合酶、细胞系、人体试验、耐药等为主要关联热点。
（所有研究的热点图）
为了更清楚地了解美国正在进行的研究，我们只是针对目前在研的98项课题进行分析。可以看到，HIV感染、环状DNA、细胞系、DNA聚合酶、长期医疗、CD8 T细胞等是目前最受关注关键词和研究热点。主持课题最多的大学包括约约翰霍普金斯大学、阿拉巴马大学伯明翰分校、南加州大学、圣路易斯大学、加州大学旧金山分校等。对于希望来美国做博士后或者访问学者的同仁，这可能是好的选择。可以看到，南加州大学的Ou, J.-H. James、阿尔巴尼医学院的Robek, Michael，伊利诺伊大学芝加哥分校的Mclachlan, Alan等目前主持课题最多。在我们昨天由Zhu, Yonghong博士撰写的乙肝治疗综述中，Dr. Zhu指出，慢性乙型肝炎之所以难以达到“功能性治愈”，就在于HBV 在感染的肝细胞中持续存在有活性的cccDNA。为此，我们特意检索了美国针对“cccDNA”的科研项目，也许对正在探索治愈乙肝的科学家们有所启发。
检索结果显示，2013年即资助了一项关于“发现cccDNA”的研究DISCOVERY OF ANTI-HBV CCCDNA COMPOUNDS FROM A UNIQUE NATURAL PRODUCTS COLLECTION。其后，于2016年开始资助项目增多，资助项目包括：2015年起，MECHANISMS FOR CCCDNA FORMATION AND PERSISTENCE
2016年起，MOLECULAR MECHANISMS OF HBV CCCDNA FORMATION2016年起，MECHANISMS FOR CCCDNA FORMATION AND PERSISTENCE2017年起，DEVELOPMENT OF AN ASSAY FOR QUANTITATIVE DETECTION OF HBV CCCDNA2017年起，DEVELOPMENT OF AN HTS ASSAY FOR DISCOVERY OF HBV CCCDNA INHIBITORS2018年起，HEPATITIS B VIRUS CCCDNA2020年起，EPIGENETIC REGULATION OF HBV CCCDNA TRANSCRIPTION我们分享在研3项cccDNA项目的课题设计思路，希望对同仁们有所启发。EPIGENETIC REGULATION OF HBV CCCDNA TRANSCRIPTIONProject Summary / Abstract:This project aims at elucidating the mechanisms underlying epigenetic regulation of hepatitis B virus (HBV) covalently closed circular (ccc) DNA transcription in hepatocytes, focusing on viral X protein (HBx)- mediated epigenetic regulation of HBV cccDNA with involvement and fine balancing of host epigenetic modulators. HBV cccDNA is essential to the virus life cycle, its complete elimination or inactivation during chronic infection is considered critical to a cure but has not been achieved by current antivirals. HBV cccDNA exists in the cell nucleus as an individual minichromosome decorated with histones and non-histone proteins. Elucidating the mechanisms of chromatin compactization of cccDNA and principles of epigenetic regulation of cccDNA episome in its interplay with host factors could allow us to elaborate new antiviral strategies for addressing the unmet clinical need. Among the limited number of HBV-encoded proteins, the viral regulatory protein HBx serves as a multifunctional transactivator of the viral and cellular promoters and has been proven to be a potent epigenetic modifying factor in HBV-infected livers. To further address the role of HBx in cccDNA transcription, we have developed a pair of inducible cccDNA reporter stable cell lines with and without HBx expression, namely HepBHAe82 and HepBHAe∆x67. While both cell lines are able to produce comparable level of cccDNA regardless of the presence or absence of HBx, the cccDNA in HepBHAe∆x67 cells is epigenetically silenced. The HBx-dependent cccDNA transcription has also been recapitulated in wildtype and HBx-minus HBV infected hepatocytes. In this project, by making use of these experimental systems, we will systematically characterize the epigenetic profile variations between transcriptionally active and inactive cccDNA (Aim 1), map the interaction of cccDNA minichromosome with host chromosomes (Aim 2), and identify host epigenetic modulators that regulate cccDNA transcription through comparative proteomic approach, followed by functional validation (Aim 3). In Aim 3, we have already identified HMGB1 as a novel host restriction factor for cccDNA, and will further elucidate the mechanism of HMGB1-mediated epigenetic repression of cccDNA transcription and the interplay between HBx and HMGB1 in cccDNA activation. The accomplishment of this project will shed more light on the cccDNA epigenetics, and provide novel antiviral targets for development of therapeutics that epigenetically silence cccDNA to achieve a functional cure of chronic hepatitis B.MECHANISMS OF HEPATITIS B VIRUS CCCDNA FORMATIONProject Summary / Abstract:Project summary Chronic hepatitis B virus (HBV) infection results in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which depends on elusive host factors for its generation. Using a yeast extract screen, we identified five core components of lagging strand synthesis –PCNA, the replication factor C (RFC) complex, DNA polymerase δ (POLδ), FEN-1, and DNA ligase 1 (LIG1) – as essential for cccDNA formation. We reconstituted cccDNA formation with purified human homologs, establishing these as a minimal set of factors necessary and sufficient for cccDNA formation. We further demonstrated that inhibiting POLδ significantly diminishes cccDNA formation. In this proposal, we will build on these findings to determine the precise kinetics of cccDNA formation, delineating the role of each factor at every step of the repair process. In understanding the dynamics of rcDNA to cccDNA repair, we can identify potential rate-limiting steps that could be novel therapeutic targets for disrupting cccDNA formation and maintenance. Using a series of innovative techniques in both cell culture and mouse model systems, we will be able to test our findings in physiologically relevant platforms that will strengthen the impact of our data. Factors found to be critical for rc- to cccDNA conversion will be disrupted in these systems by a degron-mediated approach that will allow for fine-tuned control of expression to alleviate any potential cytotoxicity. We can then monitor the effect of each factor in turn on cccDNA formation or the maintenance of established cccDNA pools in chronically infected cells. To increase the resolution of such studies, we will also examine at the single-cell level how the expression levels of a given factor correlate with that of cccDNA. Altogether, these data will give us a far more comprehensive view of this process critical to the persistence of HBV in chronically infected individuals.Project Summary / Abstract:Hepatitis B virus chronically infects approximately 300 million people worldwide. All are at significantly increased risk of developing hepatocellular carcinoma. Although virus replication can be blocked by therapy with nucleoside analogs, infected hepatocytes are not cured, because covalently closed circular DNA (cccDNA), the template for transcription of viral RNAs persists in infected cells. So far, therapeutic strategies to prevent cccDNA formation or functionally inactivate or even destroy cccDNA within infected hepatocytes are lacking. The purpose of this application is to investigate four problems pertinent to cccDNA biology: identification of the enzymes required for cccDNA formation, elucidation of the exact mechanism for cccDNA formation, investigating how cccDNA is distributed to daughter cells during regeneration of hepatocytes and determining the physical organization of cccDNA in infected hepatocytes. Research on the mechanism of cccDNA formation could reveal the identity of cellular DNA repair enzymes that in turn, could be become targets for novel antiviral therapies. Investigations on the physical location of cccDNA in nuclei of infected cells could shed light on how cccDNA is maintained and distributed to daughter cells following cell division, and how it is eventually lost during spontaneous recovery from natural HBV infections. A better understanding of these processes is deemed essential for the development of novel strategies to cure chronic hepatitis B (CHB). Aim 1. Mechanism for cccDNA synthesis. We will identify still elusive cellular enzymes responsible for the conversion of rc to cccDNA. Candidates are DNA ligase(s) and endonuclease(s), required for the processing and joining of the 5’ and 3’ ends of the two rcDNA strands. Gene knockout cells will be used to determine how rcDNA is converted into cccDNA. In addition, we propose the development of a novel screening assay for identification of host genes required for HBV infection and cccDNA formation. Aim 2. cccDNA fate during cell division and nuclear localization. This aim builds on our ability to visualize cccDNA in metaphase and interphase nuclei by fluorescence in situ hybridization (FISH). The goal is to determine the distribution of cccDNA in DHBV and HBV producing cells and investigate how cccDNA is distributed to daughter cells under normal conditions and in the presence of cytokines. Experiments are designed to investigate the nuclear localization of functional and transcriptionally silenced cccDNA. To address these aims, we will rely on an experimental approach that builds on our research efforts during the past two years leading to cell culture platforms permitting HBV infections of HepG2 cells in combination with CRISPR/Cas9 gene knockout technology. In addition, the aims build on our recent success in developing methods permitting detection of cccDNA by FISH.这个摘要不仅仅提供了精彩的课题设计思路，更是撰写基金摘要的范本。
小结：
1，尽管美国乙型肝炎病毒（HBV）感染率非常低，但美国仍然在乙肝研究领域给予了持续稳定的基金资助。累计资助2,059项HBV研究，资助基金超过5亿美元。
2，目前美国有98项在研的HBV，关注热点是合并HIV感染、环状DNA、细胞系、DNA聚合酶、长期医疗、CD8 T细胞等。
3，主持课题最多的大学包括约约翰霍普金斯大学、阿拉巴马大学伯明翰分校、南加州大学、圣路易斯大学、加州大学旧金山分校等。对于有意向来做博士后或者访问学者的同仁，这些是不错的选择。
4，主持课题最多的PI包括南加州大学的Ou, J.-H. James、阿尔巴尼医学院的Robek, Michael，伊利诺伊大学芝加哥分校的Mclachlan, Alan等。
5，我们重点关注了在妨碍功能性治愈的cccDNA研究，目前有3项很有趣的研究课题。期待通过对其摘要的分享，可以给其他研究者以启发，期待能够引发最终治愈乙肝的新思维。
6，由于我们的专业知识所限，本大数据分析可能存在误差；且仅为学术交流用。也期待各位专家点评及提出改进意见。
后台回复“美国基金”获得更多对美国在研基金热点的解读；回复“大数据”获得针对发表的论文和美国基金的热点解读；支持按照科室检索，如回复”传染病科“或者相关资料。
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