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

• 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
• 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

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摘要 最近，研究了表达CFTR的人和小鼠气道上皮细胞的复杂性。 通过单细胞RNA测序（scRNAseq）揭示。这些研究确定了离子细胞，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 表明离子细胞中的离子通道库是唯一适合于潜在地调节气道pH的。 然而，是否只有离子细胞有助于先天免疫和气道中的清除仍有待进一步研究。 确定;其他气道细胞类型，如纤毛细胞，表达CFTR的水平可能是 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。 深入了解气道中CFTR细胞生理学将大大提高我们对CF的理解 发病机制，同时也为CF肺病的基因治疗提供了细胞靶点。拟议 项目将侧重于了解CFTR在离子细胞和纤毛细胞中的细胞功能，以及是否 CFTR在这些细胞类型中的每一种中的表达是预防CF肺病所必需的或足够的。我们 假设将在遗传雪貂模型中进行测试，雪貂是一种更准确地反映人类CF肺的物种 比老鼠的病。这些研究是可能的，因为创造了几个新的遗传雪貂模型 其可以有条件地（在特定细胞类型中）抑制野生型（WT）背景上的CFTR表达，或 在CF背景上重新激活CFTR表达。这两种策略都使用CreERT 2技术， 能够使用荧光Cre报告基因在体内追踪靶细胞的谱系。此外，我们建议 产生一种新的遗传雪貂模型，其中CFTR在纤毛细胞中特异性过表达，以测试 其中的高水平异位表达是否足以防止CF肺病。这方面的主要目标 本研究的目的是：1）确定CFTR在离子细胞和纤毛细胞中的表达对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，和3） 确定CFTR在离子细胞和纤毛细胞中的表达在多大程度上有助于调节 气道表面液体（ASL）的疾病相关特征（即体积、抗菌活性、氯化物和 碳酸氢盐转运和pH）和粘膜纤毛清除。这些目标中的每一个都利用了 Engelhardt实验室对转基因雪貂进行基因工程改造，以暂时调节CFTR的表达， 特定的细胞类型，并在体内和体外测试这些模型的CF相关功能终点。的 这项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CFTR在气道中的功能和CF肺发病机制有关的困难的细胞生物学问题。本研究 预计将显著提高该领域开发CF肺病有效基因疗法的能力。