Human TfR1-expressing hamsters to model New World arenaviral hemorrhagic fever

表达人类 TfR1 的仓鼠用于模拟新世界沙病毒出血热

基本信息

批准号：
10375486
负责人：
Brian B. Gowen
金额：
$ 7.3万
依托单位：
UTAH STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-19 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10375486
关键词：
Americas Animal Model Antiviral Agents Antiviral Therapy Arenavirus Arenavirus Infections Argentinian Hemorrhagic Fever Blood Coagulation Disorders Blood Vessels Blood specimen Body Weight CRISPR/Cas technology Clinical Complement Complementary DNA Complex Development Disease Drug or chemical Tissue Distribution Ebola virus Encephalitis Engineering Ensure Evaluation Genes Genome Genomics Goals Guide RNA Hamster Cell Line Hamsters Hemorrhage Human Infection Injections Junin virus Knock-in Knock-in Mouse Lead Life Marburgvirus Measurable Measurement Mediating Mesocricetus auratus Modeling Mucous Membrane Mus National Security Normal tissue morphology Oncology Open Reading Frames Pathogenesis Pathogenicity Patients Pattern Peptides Petechiae Physiological Population Predisposition Procedures Public Health Records Research Research Personnel Research Project Grants Resources Ribonucleoproteins Risk Rodent Severity of illness Symptoms Syndrome TFRC gene Temperature Terminator Codon Testing Therapeutic Intervention Time Transgenic Organisms Vaccine Therapy Viral Hemorrhagic Fevers Viral Load result Viremia Virus Virus Diseases Virus Replication Zoonoses base cancer therapy design drug candidate experience human disease improved outcome innovation mouse transferrin receptor 1 neutrophil novel receptor success therapeutic vaccine transmission process

项目摘要

PROJECT SUMMARY Mammarenaviruses are endemic in rodent populations worldwide and their zoonotic transmission can lead to a severe life-threatening hemorrhagic fever (HF) syndrome. In the Americas, five New World mammarenaviruses (NWAs) are known to cause HF. In the absence of FDA-licensed vaccines or antiviral therapies, these viruses pose a significant public health risk and a threat to national security. Research to understand NWA pathogenesis and develop effective countermeasures is severely limited by the lack of small-animal models that faithfully mirror human disease. We recently demonstrated that transgenic expression of human transferrin receptor 1 (huTfR1), the known cellular receptor used by the pathogenic NWAs, confers susceptibility in mice to lethal disease following challenge with the Junin arenavirus (JUNV), the agent of Argentine HF. However, the mice do not manifest signs of hemorrhagic disease that are prominent features of severe cases of NWA HF in humans. We and others have shown that golden Syrian hamsters infected with related nonpathogenic NWAs that do not use huTfR1 develop a severe HF-like syndrome with many of the cardinal features of hemorrhagic disease, including coagulopathy, extensive petechia, bleeding from the oronasal mucosal region and vascular leak. Moreover, we recently showed that expression of huTfR1 in hamster cell lines significantly augments JUNV infection. Thus, we hypothesize that hamsters engineered to express huTfR1 will be susceptible to JUNV infection, resulting in a HF-like syndrome more representative of human disease. To explore this hypothesis, we will pursue the following Specific Aims: Aim 1. Develop a huTfR1 knock-in (KI) golden Syrian hamster line. We will design and test single guide (sg)RNAs targeting the 3’ end of the hamster TfR1 gene for insertion of the huTfR1 open reading frame. By appending the huTfR1 cDNA via a T2A peptide linker immediately before the stop codon of the hamster TfR1 gene, we will ensure expression of huTfR1 at physiological levels and with normal tissue distribution. The huTfR1 hamster line will be generated by a well-established pronuclear injection procedure with the Cas9/sgRNA ribonucleoprotein complex and the huTfR1 cDNA donor construct. Aim 2. Evaluate the susceptibility of the huTfR1 hamster to JUNV infection. Wild-type and huTfR1 KI hamsters will be challenged with JUNV to assess differences in viral replication and development of disease. We anticipate that expression of huTfR1 will boost viral replication leading to a HF-like syndrome in the hamsters. Disease parameters evaluated daily will include body weight, temperature and clinical disease signs including petechia and mucosal bleeding. Viral loads will be determined from blood samples taken one week after JUNV challenge, a point at which huTfR1 hamsters are expected to develop signs of disease and have measurable viremia. The ultimate goal of this project is to produce a novel hamster model of NWA HF to support development of host-directed therapies that would complement direct-acting antivirals to improve outcomes in patients suffering from severe disease caused by pathogenic NWAs.

项目概要乳房病毒在全世界啮齿类动物中流行，其人畜共患传播可导致在美洲，有五种新世界乳腺病毒导致严重危及生命的出血热 (HF) 综合征。已知 NWA 会导致心力衰竭，在没有 FDA 许可的疫苗或抗病毒疗法的情况下，这些病毒会引起心力衰竭。对了解 NWA 发病机制的研究构成重大公共卫生风险和威胁。由于缺乏忠实反映的小动物模型，制定有效的对策受到严重限制我们最近证明了人转铁蛋白受体 1 (huTfR1) 的转基因表达，致病性 NWA 使用的已知细胞受体使小鼠对致命疾病具有易感性然而，在用胡宁沙粒病毒（JUNV）（阿根廷心力衰竭的病原体）攻击后，小鼠却没有这样做。出血性疾病的体征是人类严重 NWA 心力衰竭病例的显着特征。和其他人已经表明，金色叙利亚仓鼠感染了相关的非致病性 NWA，但不使用 huTfR1 会产生严重的心力衰竭样综合征，具有出血性疾病的许多主要特征，包括凝血障碍、广泛瘀点、口鼻粘膜区域出血和血管渗漏。最近表明，仓鼠细胞系中 huTfR1 的表达显着增强了 JUNV 感染。我们发现，经过基因改造以表达 huTfR1 的仓鼠将容易受到 JUNV 感染，从而导致一种更能代表人类疾病的心衰样综合征为了探索这一假设，我们将追求具体目标如下：目标 1. 开发 huTfR1 敲入 (KI) 金色叙利亚仓鼠系。并测试针对仓鼠 TfR1 基因 3' 端的单引导 (sg)RNA 以插入 huTfR1 开放通过在终止密码子之前立即添加 huTfR1 cDNA 的 T2A 肽接头。仓鼠 TfR1 基因，我们将确保 huTfR1 在生理水平和正常组织中表达 huTfR1 仓鼠系将通过完善的原核注射程序产生。 Cas9/sgRNA 核糖核蛋白复合物和 huTfR1 cDNA 供体构建体目标 2. 评估 huTfR1 仓鼠对 JUNV 感染的易感性将受到挑战。与 JUNV 一起评估病毒复制和疾病发展的差异。 huTfR1 会促进病毒复制，导致仓鼠出现类似 HF 的疾病参数。每日评估包括体重、体温和临床疾病体征，包括瘀点和粘膜出血量将通过 JUNV 攻击后一周采集的血液样本来确定，这一点为 huTfR1 仓鼠预计会出现疾病迹象并具有可测量的病毒血症。该项目的目标是生产一种新颖的 NWA HF 仓鼠模型，以支持主机导向的开发补充直接作用抗病毒药物的疗法，以改善患有严重疾病的患者的预后由致病性 NWA 引起的疾病。