Conducting Airway Cellular Targets Required for Complementation of CF Lung Disease

传导补充 CF 肺病所需的气道细胞靶点

基本信息

批准号：
10470338
负责人：
JOHN F ENGELHARDT
金额：
$ 48.65万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10470338
关键词：
Address Adopted Airway Fibrosis Amphotericin B Anions Anti-Bacterial Agents Area Bicarbonates Cell physiology Cells Cellular biology Chlorides Complement Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Development Disease Disease Progression Duct (organ) structure Ectopic Expression Epithelial Cells Ferrets Genetic Genetic Engineering Gland Goals Half-Life Human In Vitro Inflammation Ion Channel Knowledge Laboratories Liquid substance Lung Mediating Messenger RNA Modeling Molecular Movement Mucociliary Clearance Mus NON Mouse Natural Immunity Pathogenesis Phenotype Process Property Pulmonary Cystic Fibrosis Regulation Reporter Research Surface Technology Testing Transgenic Organisms absorption airway epithelium airway surface liquid cell type cellular targeting cystic fibrosis airway defined contribution detection limit disease phenotype epithelial Na+ channel gain of function gene complementation gene replacement gene therapy genetic approach in vivo loss of function overexpression porcine model prevent pulmonary function restoration single-cell RNA sequencing synergism

项目摘要

Project-3 Summary Complexities in CFTR-expressing epithelial cells of the conducting human and mouse airway were recently revealed by single-cell RNA sequencing (scRNAseq). These studies identified the ionocyte, an infrequent cell type that expresses the majority of CFTR in the proximal airway and submucosal gland ducts. They also showed that the repertoire of ion channels in the ionocyte is uniquely suited to potentially regulate airway pH. However, whether only ionocytes contribute to innate immunity and clearance in the airways remains to be determined; it is possible that other airway cell types, such as ciliated cells, express CFTR at levels that are below the detection limits of scRNAseq yet are functionally important in the pathogenesis of CF lung disease. Deeper knowledge of CFTR cellular physiology in the airway will greatly enhance our understanding of CF pathogenesis, while also informing the cellular targets for gene therapy of CF lung disease. The proposed project will focus on understanding the cellular functions of CFTR in ionocytes and ciliated cells, and whether CFTR expression in each of these cell types is required or sufficient to prevent CF lung disease. Our hypotheses will be tested in genetic ferret models, a species that more accurately reflects human CF lung disease than mice. These studies are possible because of the creation of several new genetic ferret models that can conditionally (in specific cell types) inactivate CFTR expression on a wild-type (WT) background or reactivate CFTR expression on a CF background. Both of these strategies use CreERT2 technologies and enable lineage tracing of the targeted cells in vivo using a fluorescent Cre-reporter. Additionally, we propose to generate a new genetic ferret model in which CFTR is overexpressed specifically in ciliated cells, to test whether high-level ectopic expression therein is sufficient to protect from CF lung disease. Key goals of this project are to: 1) define the contributions of CFTR expression in ionocytes and ciliated cells to CF lung pathogenesis, 2) determine the half-lives of ionocytes and ciliated cells in the CF and non-CF airway, and 3) determine the extent to which CFTR expression in ionocytes and ciliated cells contributes to the regulation of disease-relevant features of the airway surface liquid (ASL) (i.e. volume, antibacterial activity, chloride and bicarbonate transport, and pH) and to mucociliary clearance. Each of these goals draws on the unique ability of the Engelhardt laboratory to genetically engineer transgenic ferrets to temporally regulate CFTR expression in specific cell types and to test these models for CF-relevant functional endpoints in vivo and in vitro. The proposed study is the first to use state-of-art functional genetic approaches in a non-mouse species to tackle difficult cell biology questions relating to CFTR function in the airway and CF lung pathogenesis. This research is expected to significantly enhance the field's ability to develop effective genetic therapies for CF lung disease.

项目 3 总结最近，人类和小鼠气道表达 CFTR 的上皮细胞的复杂性通过单细胞 RNA 测序 (scRNAseq) 揭示。这些研究鉴定了离子细胞，一种罕见的细胞在近端气道和粘膜下腺管中表达大部分 CFTR 的类型。他们还研究表明，离子细胞中的离子通道库非常适合潜在地调节气道 pH 值。然而，是否只有离子细胞有助于先天免疫和气道清除仍有待研究。决定;其他气道细胞类型（例如纤毛细胞）可能以以下水平表达 CFTR：低于 scRNAseq 的检测限，但在 CF 肺病的发病机制中具有重要的功能。更深入地了解气道 CFTR 细胞生理学将极大地增强我们对 CF 的理解发病机制，同时还为 CF 肺病基因治疗的细胞靶点提供信息。拟议的该项目将重点了解 CFTR 在离子细胞和纤毛细胞中的细胞功能，以及是否这些细胞类型中的每一种细胞中的 CFTR 表达对于预防 CF 肺病都是必需的或足够的。我们的假设将在遗传雪貂模型中进行测试，该物种能够更准确地反映人类 CF 肺病比老鼠多。这些研究之所以成为可能，是因为几种新的遗传雪貂模型的创建可以有条件地（在特定细胞类型中）使野生型 (WT) 背景上的 CFTR 表达失活，或在 CF 背景上重新激活 CFTR 表达。这两种策略都使用 CreERT2 技术使用荧光 Cre 报告基因对体内目标细胞进行谱系追踪。此外，我们建议生成一个新的遗传雪貂模型，其中 CFTR 在纤毛细胞中特异性过度表达，以测试其中的高水平异位表达是否足以预防 CF 肺病。本次活动的主要目标项目目标是： 1) 确定离子细胞和纤毛细胞中 CFTR 表达对 CF 肺的贡献发病机制，2) 确定 CF 和非 CF 气道中离子细胞和纤毛细胞的半衰期，以及 3) 确定离子细胞和纤毛细胞中 CFTR 表达对调节的程度气道表面液体 (ASL) 的疾病相关特征（即体积、抗菌活性、氯离子和碳酸氢盐转运和 pH）以及粘液纤毛清除。这些目标中的每一个都利用了独特的能力 Engelhardt 实验室对转基因雪貂进行基因改造，以暂时调节 CFTR 的表达特定细胞类型，并在体内和体外测试这些模型的 CF 相关功能终点。这拟议的研究是第一个在非小鼠物种中使用最先进的功能遗传方法来解决与气道 CFTR 功能和 CF 肺发病机制相关的细胞生物学难题。这项研究预计将显着增强该领域开发针对 CF 肺病的有效基因疗法的能力。