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

• 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
• 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

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.

项目摘要 在全球啮齿动物种群中，乳腺癌病毒是特有的，它们的人畜传播可能会导致 严重的杀人生命出血热（HF）综合征。 （NWA）在没有FDA许可的疫苗或抗病毒疗法的情况下引起HF。 构成了重大的公共卫生风险和对国家安全的威胁。 并且发展有效的对策受到缺乏忠实反映的小动物模型的限制 人类疾病。 致病性NWA使用的已知地窖受体，赋予小鼠致命疾病的敏感性 在与阿根廷HF的特工Junin Arenavirus（Junv）挑战之后。 明显的出血性疾病的迹象表明，NWA HF的严重Cass在人类中 其他人则表明，金叙利亚仓鼠与相关的非人性化NWAT无关 HUTFR1发展出严重的HF样综合征，具有许多出血性疾病的基本特征，包括 凝血病，广泛的petechia，来自Oronasal粘膜区域的出血和血管泄漏 最近表明，HutFR1在仓鼠细胞lin中的表达显着增强。 我们假设仓鼠发动机表达HUTFR1将容易受到JUNV感染的影响，恢复 HF样综合征对人类疾病的抑制作用。 遵循特定的目标：AIM 1。开发HutFR1敲击（Ki）金叙利亚仓鼠 和测试单引导（SG）RNA靶向仓鼠TFR1基因的3'端以插入HUTFR1打开 通过T2A肽接头添加HUTFR1 cDNA，在终止密码子之前 仓鼠TFR1基因，我们将确保HUTFR1在正常的生理水平上表达 分布式。 CAS9/SGRNA核糖核蛋白复合物和HUTFR1 cDNA供体构建体。 HUTFR1仓鼠对JunV感染的敏感性。 随着JUNV评估病毒复制和疾病发展的差异。 HUTFR1的of将增强病毒复制，从而导致仓鼠中的HF样综合征。 评估的Daly将包括体重 出血。 HUTFR1仓鼠有望产生疾病的迹象，并具有可测量的病毒性 这样做的目标是生产新型NWA HF的仓鼠模型，以支持宿主定向的开发 可以补充直接作用抗病毒药的疗法，以改善严重患者的预后 病原NWA引起的疾病。