Defining mechanisms of Zika-virus associated microcephaly: cell population dynamics and gene expression in infected human cerebral organoids and neural progenitor cells

寨卡病毒相关小头畸形的定义机制：受感染的人脑类器官和神经祖细胞的细胞群动态和基因表达

• 批准号: 10455429
• 负责人: Karen Emily Ocwieja
• 金额: $ 19.49万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-23 至 2022-09-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10455429
• 关键词: Address; Advisory Committees; Africa; African; American; Americas; Apoptosis; Area; Asian; Autopsy; Biology; Blood Circulation; Boston; Brain; CRISPR/Cas technology; Cell Culture Techniques; Cell Cycle; Cell Death; Cell Line; Cell division; Cells; Cerebrum; Childhood; Communicable Diseases; Complex; Computational Biology; Cytomegalovirus; Defect; Development; Developmental Biology; Disease Outbreaks; Educational workshop; Engineering; Epidemic; Experimental Models; Fetal Development; Flavivirus; Foundations; Future; Gene Expression; Gene Expression Profile; Genes; Genetic Translation; Genome; Goals; Growth; High-Throughput Nucleotide Sequencing; Human; Infection; Institutes; International; Investigation; Knowledge; Leadership; Learning; Measures; Medical; Mentorship; Methods; Microcephaly; Micronesia; Microscopic; Mitosis; Modeling; Molecular; Neuraxis; Neurons; Normal tissue morphology; Organoids; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Pediatric Hospitals; Physicians; Poly(A) Tail; Polynesian; Population; Population Dynamics; Positioning Attribute; RNA; Regulation; Reporting; Research; Research Personnel; Research Training; Resources; Role; Rubella virus; Science; Scientist; Severities; Severity of illness; Specimen; Syndrome; Techniques; Technology; Teratogens; Time; Tissue Culture Techniques; Tissue Model; Tissues; Training; Training Programs; Translating; Viral Genome; Viral Pathogenesis; Virulence; Virus; Virus Replication; Vocational Guidance; Work; ZIKV infection; Zika Virus; career; career development; cell immortalization; cell type; congenital infection; congenital zika syndrome; cost; fetal; human embryonic stem cell; human stem cells; improved; insight; medical schools; mouse model; nerve stem cell; neurogenesis; novel; novel therapeutics; pathogen; polyadenylated messenger RNA; premature; prevent; response; ribosome profiling; single cell sequencing; single-cell RNA sequencing; stem cell derived tissues; stem cells; supportive environment; symposium; three dimensional structure; tool; transcriptome; transcriptomics; translatome; virology

PROJECT SUMMARY/ABSTRACT Zika virus swept across the Americas in 2015-16, and it was during this epidemic that the teratogenic consequences of congenital Zika infection were first described. Despite extensive research, it remains unknown how Zika infection causes microcephaly, or whether this pathology is unique to recent strains, Two new tools will facilitate discovery in this area. First, we and others have shown that the human stem cell derived cerebral organoid is a tractable neurodevelopmental model in which to study Zika infection. Second, we have demonstrated that single cell sequencing techniques that enrich for poly-adenylated mRNAs can identify flavivirus-infected cells – an unexpected result given flavivirus genomes do not have poly(A) tails. The goal of this study is to determine the mechanisms by which Zika virus disrupts fetal brain development, building on these technical advances. We will employ single cell RNA-sequencing to identify and compare cellular populations in organoids over time, in the presence and absence of Zika virus. This will allow us to distinguish pathogenic disruptions in a) stem cell abundance, b) cell division, and c) differentiation. We will use single cell RNA-sequencing and ribosome profiling to define gene expression responses to Zika infection in neural progenitor cells, thought to be the target of Zika virus in the fetal brain. By comparing viruses of varying pathogenicity in these studies, we will identify differences in host gene expression and viral replication associated with disease severity. The proposed study will address a major unresolved problem in the field of Zika virus pathogenesis, contribute broadly to our understanding of cerebral development, and mature cutting edge technologies for the investigation of questions at the interface of infection and development. The project will be developed under the mentorship of Dr. Lee Gehrke, a leader and expert in the field of RNA virology with a background in developmental biology. Additional scientific and career guidance will be provided by a scientific advisory committee composed of experts in virology, single-cell sequencing, stem-cell derived tissue models, and pediatric infectious disease pathogenesis. The training program will include coursework in computational biology, training in molecular and tissue culture techniques, workshops in leadership, and didactic learning from local seminars as well as national and international conferences. Research and training will occur at the MIT Institute for Medical Engineering and Science, and at Boston Children’s Hospital at the Harvard Medical School. Together, MIT and Harvard Medical School afford extensive resources and expertise in all aspects of the proposed research. Boston Children’s Hospital is a supportive environment committed to providing 85% protected time for this research, and offers workshops in career development and leadership to prepare early-stage investigators for independence. The project will build on the candidate’s background in virology and high-throughput sequencing, allow her to mature as a physician-scientist, and position her to achieve her goal of an independent research career in pediatric infectious disease pathogenesis.

项目概要/摘要 寨卡病毒于2015-16年席卷美洲，正是在这次流行期间，致畸的病毒 首先描述了先天性寨卡病毒感染的后果。尽管进行了广泛的研究，但它仍然 未知寨卡病毒感染如何导致小头畸形，或者这种病理学是否是最近的菌株所独有的，两个 新工具将促进这一领域的发现。首先，我们和其他人已经证明人类干细胞 衍生的大脑类器官是一种易于处理的神经发育模型，可用于研究寨卡病毒感染。第二， 我们已经证明，富集聚腺苷酸化 mRNA 的单细胞测序技术可以 识别黄病毒感染的细胞——这是一个意想不到的结果，因为黄病毒基因组没有多聚（A）尾。这 这项研究的目标是确定寨卡病毒破坏胎儿大脑发育的机制， 建立在这些技术进步的基础上。我们将采用单细胞 RNA 测序来识别和比较 在存在和不存在寨卡病毒的情况下，随着时间的推移，类器官中的细胞群。这将使我们能够 区分 a) 干细胞丰度、b) 细胞分裂和 c) 分化的致病性破坏。我们将 使用单细胞 RNA 测序和核糖体分析来定义对寨卡病毒感染的基因表达反应 神经祖细胞被认为是寨卡病毒在胎儿大脑中的目标。通过比较病毒 在这些研究中不同的致病性，我们将确定宿主基因表达和病毒复制的差异 与疾病严重程度有关。拟议的研究将解决该领域尚未解决的主要问题 寨卡病毒发病机制，广泛有助于我们了解大脑发育和成熟切割 用于研究感染和发展界面问题的边缘技术。 该项目将在RNA领域的领导者和专家Lee Gehrke博士的指导下开发 具有发育生物学背景的病毒学。将提供额外的科学和职业指导 由病毒学、单细胞测序、干细胞衍生专家组成的科学咨询委员会 组织模型和儿科传染病发病机制。培训计划将包括课程作业 计算生物学、分子和组织培养技术培训、领导力研讨会，以及 从当地研讨会以及国家和国际会议中进行教学学习。研究与培训 将在麻省理工学院医学工程与科学研究所和波士顿儿童医院举行 哈佛医学院。麻省理工学院和哈佛医学院共同提供广泛的资源和专业知识 在拟议研究的各个方面。波士顿儿童医院致力于营造一个支持性环境 为这项研究提供 85% 的受保护时间，并提供职业发展和领导力研讨会 为早期调查人员的独立做好准备。该项目将建立在候选人的背景之上 在病毒学和高通量测序方面的专业知识，使她能够成为一名成熟的医师科学家，并使她能够 实现她在儿科传染病发病机制方面独立研究职业的目标。