Modeling early SARS-CoV-2 pathogenesis in human lung organoids and slice cultures

项目摘要 COVID-19仍然是一场持续的全球健康危机，部分原因是新出现的变种。基本问题 关于SARS-CoV-2感染驱动下肺炎症的机制， 由于缺乏体外感染模型。更好地理解变异生物学和免疫途径 参与早期SARS-CoV-2感染可能为开发新的治疗策略提供见解。 抗病毒细胞因子I型干扰素及其下游效应与COVID-19有关。 SARS-CoV-2可能抑制或促进肺中某些细胞类型的干扰素效应， 病毒变种之间的联系我的初步工作表明，类器官和肺切片培养可用于研究 这些早期事件在SARS-CoV-2感染，并确定巨噬细胞作为一种细胞类型，激活 干扰素途径感染的文化。因此，我建议使用定量PCR，单细胞RNA测序， 和流式细胞术来研究病毒变体，并深入分析病毒和宿主基因和蛋白的变化 感染时的表达。我将研究高度相关的细胞类型，它们可以以类似的方式相互作用， 在体内肺的方式。我将研究干扰素刺激的基因反应，并确定细胞类型， 其被调节或将是治疗干预的良好靶点。这些知识将至关重要 我们抗击COVID-19疫情的努力。 我目前是斯坦福大学传染病系凯瑟琳·布利什博士实验室的研究员 我正在被提升为全职教练。斯坦福大学提供了一个 杰出的科学环境，在那里所有必要的资源，这个项目的成功， 我的长期目标是成为一名独立的物理学家，科学家，研究重点是 呼吸道病毒发病机制我的目标是建立一个研究计划，重点是建立模型， 新出现的原发性肺组织感染，目的是开发新的治疗方法。 为了实现这一目标，我设计了一个量身定制的职业发展计划， 非正式培训。这次培训将增强我在肺生物学、免疫学和测序方面的专业知识 分析.这些主题的非正式培训将由我的导师凯瑟琳·布利什博士和其他人提供。 我的顾问委员会，卡尔文郭博士，马克·克拉斯诺博士，苏珊·霍姆斯博士和本·平斯基博士。与他们的 指导，我将完成拟议的项目，提交研究手稿，获得进一步的资金，并获得 独立的研究职位。

PROJECT SUMMARY COVID-19 remains an ongoing global health crisis, in part due to emerging variants. Fundamental questions remain about the mechanisms by which SARS-CoV-2 infection drives inflammation in the lower lung, in part due to the lack of in vitro infection models. Better understanding of variant biology and the immune pathways involved in early SARS-CoV-2 infection could offer insights for the development of novel therapeutic strategies. The antiviral cytokines type I interferons and their downstream effects have been implicated in COVID-19. SARS-CoV-2 may inhibit or promote interferon effects in some cell types in the lung, and this may differ between viral variants. My preliminary work shows that organoids and lung slice cultures can be used to study these early events in SARS-CoV-2 infection and identifies macrophages as a cell type that activates the interferon pathway in infected cultures. Thus, I propose using quantitative PCR, single-cell RNA sequencing, and flow cytometry to study viral variants and to deeply profile the changes to viral and host gene and protein expression during infection. I will study highly relevant cell types that can interact with each other in a similar fashion to the in vivo lung. I will study the interferon stimulated gene response and identify the cell types in which it is being modulated or would be good targets for therapeutic intervention. This knowledge will be critical to our efforts to combat the COVID-19 pandemic. I am currently a fellow in Dr. Catherine Blish’s lab in the Division of Infectious Diseases at Stanford University and I am in the process of being promoted to a full time Instructor position. Stanford University offers an outstanding scientific environment, where all the resources necessary to the success of this project are made available to me. My long-term goal is to become an independent physician-scientist, with a research focus in respiratory viral pathogenesis. I aim to establish a research program focused on modeling established and emerging infections in primary lung tissue with a goal of developing new treatments. To achieve this goal, I have designed a tailored career development plan that comprises both formal and informal training. This training will enhance my expertise in lung biology, immunology, and sequencing analysis. Informal training in these topics will be provided by my mentor, Dr. Catherine Blish, and by the rest of my advisory committee, Dr. Calvin Kuo, Dr. Mark Krasnow, Dr. Susan Holmes, and Dr. Ben Pinsky. With their guidance, I will complete the proposed project, submit research manuscripts, obtain further funding, and obtain an independent research position.