Transposon Mutagenesis for Host-Target and Drug Discovery in Infectious Disease

传染病宿主靶标和药物发现的转座子诱变

基本信息

批准号：
8883359
负责人：
Adam Lacy-Hulbert
金额：
$ 49.99万
依托单位：
BENAROYA RESEARCH INST AT VIRGINIA MASON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8883359
关键词：
Acute Antiviral Agents Antiviral resistance Arenavirus Bioinformatics Bioterrorism Candidate Disease Gene Cell Death Cell Line Cells Chronic Communicable Diseases Data Drug resistance Ebola Hemorrhagic Fever Ebola virus Filovirus Frankfurt-Marburg Syndrome Virus Gene Expression Generations Genes Genetic Screening Goals Healthcare Host resistance Immune Immune system Immunity Individual Infection Infectious Agent Integration Host Factors Lead Libraries Mediating Mutagenesis Mutation Outcome Pathway interactions Pattern recognition receptor Phase RNA Viruses Resistance Signal Pathway Site System Testing Therapeutic Therapeutic Intervention Up-Regulation Variant Vesicular stomatitis Indiana virus Viral Viral Hemorrhagic Fevers Virus Virus Diseases base care burden chemokine chemotherapy combat cytokine drug discovery genome-wide mutant novel novel therapeutics prevent promoter research study resistance gene response screening small molecule success

Acute Antiviral Agents Antiviral resistance Arenavirus Bioinformatics Bioterrorism Candidate Disease Gene Cell Death Cell Line Cells Chronic Communicable Diseases Data Drug resistance Ebola Hemorrhagic Fever Ebola virus Filovirus Frankfurt-Marburg Syndrome Virus Gene Expression Generations Genes Genetic Screening Goals Healthcare Host resistance Immune Immune system Immunity Individual Infection Infectious Agent Integration Host Factors Lead Libraries Mediating Mutagenesis Mutation Outcome Pathway interactions Pattern recognition receptor Phase RNA Viruses Resistance Signal Pathway Site System Testing Therapeutic Therapeutic Intervention Up-Regulation Variant Vesicular stomatitis Indiana virus Viral Viral Hemorrhagic Fevers Virus Virus Diseases base care burden chemokine chemotherapy combat cytokine drug discovery genome-wide mutant novel novel therapeutics prevent promoter research study resistance gene response screening small molecule success

展开

项目摘要

Although both acute and chronic viral infections remain a major health care burden, the therapeutic options are limited. Some of the most concerning of the viral infections include those caused by the Filoviruses and the Arenaviruses. These RNA viruses cause severe hemorrhagic fevers that are often lethal. Early defense against all viruses is mediated by the innate immune system and the responses triggered by pattern recognition receptors that induce the cytokines and chemokines that orchestrate much of the local and systemic anti-viral defenses. However, in addition to these well- defined innate immune signaling pathways, less well-understood cell autonomous factors also contribute to protect the host from and during viral infection. Based on emerging data and our preliminary studies, we hypothesize that an important strategy of host resistance against viruses is the upregulation of `restriction factors' that prevent viral entry, restrict the ability of viruses to infect or replicate in host cells or increase the ability of cells to withstand viral-induced cytopathy. However, although these restriction factors are likely to provide a wealth of new targets for therapeutic intervention, the identities of such cell-autonomous host factors that protect against viruses are largely unknown. Here we propose to utilize and further optimize a novel unbiased screening approach using transposon mutagenesis. This strategy relies three steps: (i) piggyBac transposon mutagenesis to generate a library of mutagenized cells, (ii) selection of mutant clones resistant to viral-induced cell death and (iii) sequencing to identify transposon insertions sites and candidate genes that contribute to protect the mutant cells from the selection agent. In the R21 phase of this proposal we aim to further develop and utilize this approach for identification of antiviral restriction factors. Additionally, in the R33 phase we propose to both validate our new host targets using native virus as well as modify the transposon system for drug discovery.

尽管急性和慢性病毒感染仍然是主要的医疗保健负担，治疗选择是有限的。一些最令人关注的病毒感染包括由丝状病毒和体育症病毒引起的感染。这些RNA 病毒引起严重的出血性发作，通常是致命的。对所有人的早期防御病毒是由先天免疫系统介导的，触发的反应诱导细胞因子和趋化因子的模式识别受体许多地方和系统的抗病毒防御。但是，除了这些定义的先天免疫信号通路，不太理解的细胞自主因素还有助于保护宿主免受病毒感染的影响。基于新兴数据和我们的初步研究，我们假设宿主对病毒的抗性是防止病毒的“限制因素”的上调进入，限制病毒在宿主细胞中感染或复制的能力或提高能力细胞以承受病毒诱导的细胞质病。但是，尽管这些限制因素可能为治疗干预提供了许多新目标这种预防病毒的细胞自主宿主因素在很大程度上尚不清楚。在这里，我们建议利用并进一步优化一种新颖的无偏筛选方法使用转座子诱变。该策略依赖三个步骤：（i）Piggybac Transposon 诱变产生诱变细胞的库，（ii）选择突变克隆对病毒诱导的细胞死亡和（iii）测序的耐药性识别转座子插入有助于保护突变细胞免受选择的位点和候选基因代理人。在此提案的R21阶段，我们旨在进一步发展和利用这一点鉴定抗病毒限制因素的方法。另外，在R33阶段我们建议使用天然病毒验证我们的新宿主目标，并修改药物发现的转座子系统。