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：
8490300
负责人：
Adam Lacy-Hulbert
金额：
$ 24.89万
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 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8490300
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 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 screening 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.

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负担，但治疗选择有限。一些最令人担忧的病毒感染包括由丝状病毒和沙粒病毒引起的感染。这些 RNA 病毒会引起严重的出血热，通常是致命的。针对所有病毒的早期防御是由先天免疫系统和模式识别受体触发的反应介导的，这些受体诱导细胞因子和趋化因子，协调大部分局部和全身抗病毒防御。然而，除了这些明确的先天免疫信号通路外，不太了解的细胞自主因素也有助于保护宿主免受病毒感染和病毒感染期间的侵害。根据新出现的数据和我们的初步研究，我们假设宿主抵抗病毒的一个重要策略是上调“限制因子”，以防止病毒进入、限制病毒在宿主细胞中感染或复制的能力或增加病毒感染宿主细胞的能力。细胞抵抗病毒诱导的细胞病变。然而，尽管这些限制因素可能为治疗干预提供大量新靶标，但此类抵御病毒的细胞自主宿主因素的身份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利用转座子诱变的新型无偏筛选方法。该策略依赖于三个步骤：（i）piggyBac 转座子诱变以生成诱变细胞文库，（ii）选择对病毒诱导的细胞死亡具有抵抗力的突变克隆，以及（iii）测序以识别转座子插入位点和有助于诱变的候选基因。保护突变细胞免受选择剂的影响。在该提案的 R21 阶段，我们的目标是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，并修改转座子系统以进行药物发现。