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：
8391403
负责人：
Adam Lacy-Hulbert
金额：
$ 21.47万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8391403
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 Filovirus 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 Screening procedure 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 response small molecule success

项目摘要

DESCRIPTION (provided by applicant): 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 nfect 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. PUBLIC HEALTH RELEVANCE: Viral infections remain a major health care burden but the therapeutic options are limited. Some of the most concerning of the viral infections include those caused by the Filoviruses and the Arenaviruses. Here we propose to develop and use a novel unbiased screening approach involving transposon mutagenesis to identify new host-targets that protect from infection with viruses. Our long-term goal is to use this to help identify new therapeutic options

描述（由申请人提供）：尽管急性和慢性病毒感染仍然是主要的医疗保健负担，但治疗方案是有限的。病毒感染最令人关注的一些包括由丝病毒和体育症病毒引起的。这些RNA病毒会导致严重的出血性发作，通常是致命的。对所有病毒的早期防御是由先天免疫系统介导的，以及诱导细胞因子和趋化因子触发的反应，这些因子和趋化因子策划了许多局部和系统性的抗病毒防御。但是，除了这些定义明确的先天免疫信号传导途径外，较不熟悉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，我们假设宿主抵抗病毒的重要策略是对“限制因素”的上调，以防止病毒进入，限制病毒在宿主细胞中NFECT或复制的能力或增加细胞的能力，以承受病毒诱导的细胞质炎。但是，尽管这些限制因素可能为治疗干预提供了许多新的靶标，但这种保护病毒的细胞自治宿主因素的身份在很大程度上是未知的。在这里，我们建议使用转座子诱变利用并进一步优化一种新型的无偏筛选方法。该策略依赖三个步骤：（i）PiggyBac转座子诱变产生诱变细胞的库，（ii）选择对病毒诱导的细胞死亡具有抗药性的突变克隆和（III）测序以鉴定转座子插入部位和候选基因，从而有助于保护突变细胞免受选择剂的影响。在本提案的R21阶段，我们旨在进一步开发和利用这种方法来识别抗病毒限制因素。此外，在R33阶段，我们建议使用天然病毒验证我们的新宿主靶标，并修改转座子系统进行药物发现。公共卫生相关性：病毒感染仍然是主要的医疗保健负担，但治疗选择有限。病毒感染最令人关注的一些包括由丝病毒和体育症病毒引起的。在这里，我们建议开发和使用一种涉及转座子诱变的新型无偏筛选方法，以鉴定可保护免受病毒感染的新宿主目标。我们的长期目标是使用它来帮助识别新的治疗选择