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Characterization of degranulation regulators in human mast cells

人类肥大细胞脱颗粒调节剂的表征

基本信息

批准号：
10284390
负责人：
Stephen Joseph Galli
金额：
$ 23.62万
依托单位：
STANFORD 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：
2021-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10284390
关键词：
Affinity Allergens Allergic Disease Allergic Reaction American Asthma Atopic Dermatitis Autoimmune Diseases Avidin Binding CD34 gene CRISPR library CRISPR/Cas technology Cell Culture Techniques Cell Degranulation Cell Line Cell Separation Cell physiology Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Confocal Microscopy Coupled Data Development Disease Economic Burden Eczema Effector Cell Food Hypersensitivity Genes Genomic Library Goals Growth Factor Hay fever Health Heparin Histamine Human Hypersensitivity IL3 Gene IgE IgE Receptors Immunity Inflammation Mediators Interleukin-6 Leukotriene C4 Libraries Mediating Mediator of activation protein Modeling Mus Mutation Parasites Pathology Pathway interactions Patients Peptide Hydrolases Phenotype Phosphotransferases Prevention Process Production Prostaglandin D2 Regulation Research Resolution Rodent Role Schools Signal Pathway Signal Transduction Signal Transduction Pathway Surface Technology Therapeutic Agents Time Transfection Work base cell preparation cellular imaging confocal imaging cost crosslink cytokine design experience functional genomics health care delivery health economics human subject immunoregulation lipid mediator mast cell mastocytosis mouse model new technology novel novel therapeutic intervention pathogen peripheral blood prevent response therapy design vector

项目摘要

PROJECT SUMMARY/ABSTRACT This project’s long-term goals are to understand the regulation of human mast cell (MC) function and to elucidate the roles of MCs in human health and disease. MCs are major effector cells in IgE-associated responses (e.g., asthma, allergy, parasite immunity). These cells also may contribute to certain autoimmune disorders and in the initiation of responses to pathogens and other agents. Upon activation, MCs undergo degranulation, leading to the secretion of many mediators (including stored products, e.g., histamine, heparin and proteases), as well as the production of lipid mediators, e.g., LTC4 and PGD2, and many cytokines and growth factors. Depending on the setting, MCs can have effector and/or immunoregulatory roles. Most research investigating MC development and function has employed various mouse models. However, we know considerably less about the mechanisms that regulate the activation of human MCs. Similarly, relatively little is known about how the perturbation of signaling pathways in human MCs can contribute to MC- associated pathology. This, in turn, has hampered the design of therapeutic agents for the treatment and/or prevention of allergies and other mast cell-associated diseases in human subjects. We recently developed a technology platform employing functional genomics, coupled with high-resolution single-cell confocal imaging, which can rapidly identify regulators of degranulation in human mast cells. We now propose to use this platform to identify key regulators of IgE/FceRI-dependent signal transduction pathways in cultured, donor- derived, primary human mast cells. Specifically, we will attempt to validate, in primary human mast cells, selected major degranulation regulators previously identified in rodent mast cells. We also will use targeted arrayed human CRISPR-Cas9 libraries to identify novel (i.e., previously unidentified) regulators of IgE/FceRI- dependent signaling pathways in primary human mast cells. In addition, we will investigate the functional significance of the KIT D816V mutation detected in mastocytosis patients by using the CRISPR-Cas9-based gene editing technologies to induce/correct the same mutation in human HMC-1 cell lines and primary human mast cells. To achieve our goals, we propose two aims. In Aim 1, we will identify novel human mast cell degranulation regulators and systematically define IgE/FceRI-dependent signaling pathways in primary human mast cells. In Aim 2, we will use CRISPR-Cas9 “prime editing” to perform mutational studies of HMC- 1 human mast cell lines and primary human mast cells. Together, this work will help create a detailed model of mast cell degranulation in human mast cells and will begin to analyze how the KIT D816V mutation may influence human mast cell biology.

项目总结/文摘