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Engineering Human Bronchial Epithelial Cells for Cystic Fibrosis Cell Therapy

工程化人类支气管上皮细胞用于囊性纤维化细胞治疗

基本信息

批准号：
10544140
负责人：
Rhianna E Lee
金额：
$ 3.11万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
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：
2021-07-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10544140
关键词：
3-Dimensional Affect Air Area Autologous Bacterial Infections Basal Cell Binding Biological Biological Assay Bronchiectasis Calcium Cell Therapy Cell surface Cells Cellular translocation Chemicals Chemotaxis Chimerism Chronic Clinic Collection Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Disease Engineering Engraftment Epithelial Epithelial Cells Filtration Genetic Diseases Genotype Goals Home Homing Human Human Engineering IL8RA gene Image Immune Immune response Impairment In Vitro Incidence Inflammation Inflammatory Injury Interleukin 8A Receptor Interleukin-8 Lentivirus Light Liquid substance Lung Lung diseases Measures Mediating Messenger RNA Methods Microfluidics Modeling Movement Mucociliary Clearance Mucous body substance Mus Mutation Operative Surgical Procedures Pathway interactions Patients Persons Pertussis Toxin Pharmacotherapy Plant Roots Play Preparation Process Proteins Pseudomonas aeruginosa Quality of life Reporter Running Safety Source Speed Sterility Stimulant Surface Testing Tight Junctions Time Trachea Translating Variant Virus Western Blotting Work airway epithelium blind bronchial epithelium cell motility cell type cellular engineering cellular transduction chemokine chemokine receptor chronic infection confocal imaging digital druggable target gene correction gene replacement genetic variant improved in vivo injured airway loss of function matrigel member migration mortality neutrophil promoter receptor expression respiratory response self-renewal severe injury stem cell niche stem cells trafficking training opportunity two photon microscopy two-dimensional two-photon

项目摘要

ABSTRACT/PROJECT SUMMARY Cystic fibrosis (CF) is a genetic disease in which the cystic fibrosis transmembrane regulator (CFTR) protein is absent or dysfunctional, leading to the buildup of viscous mucous, impaired mucociliary transport, chronic infection and inflammation, and eventually mortality. The cause of CF is challenging to treat, with over 300 known disease-causing protein variants. Cell therapy offers a mutation-blind treatment option; however, cell engraftment into the highly resistive airway epithelium is ineffective and requires extensive airway injury. Approaches to facilitate cellular movement across tight junction barriers and to direct delivered cells to the stem cell niche may be an alternative. Though challenging in the cell therapy model, cellular translocation across epithelial barriers is not without biological precedent. Neutrophils readily migrate into the air space by chemotaxis, the directed migration of cells in response to a chemical stimulant. This motility is initiated when chemokine receptors on the neutrophil’s surface bind target chemokines. In the lungs, chemokines are produced by basal cells, the stem cells of the upper airways and chief residents of the stem cell niche. This process is exceptionally relevant in CF, where bacterial colonization is chronic and the immune response runs rampant. Though chemotaxis is well studied in neutrophils and other immune cells, it is unknown if artificial expression of chemokine receptors in non-immune primary cells would enable them to chemotax through tight junction barriers. Preliminary results from our lab indicate that primary human bronchial epithelial cells (HBECs) engineered to express the chemokine receptor CXCR1 chemotax in the presence of an interleukin-8 (IL8) gradient. As such, our central hypothesis is that engineering HBECs to express chemokine receptors will promote directed migration toward the chemokine source (i.e., the stem cell niche), thus improving the efficiency of cell engraftment. To test this hypothesis, CXCR1 expression will be optimized in HBECs using diverse lentiviral promoters. CXCR1 abundance will be quantified by western blot, and directional migration will be measured in two- and three-dimensional chemotaxis assays. Canonical chemotaxis pathway members will be quantified by western blot and fluorescent reporter assays. The goal of directing exogenously delivered cells toward the stem cell niche is ultimately to increase long-term cell engraftment with minimal injury requirements. Thus, the engraftment efficiency of engineered HBECs will be evaluated in vitro by quantifying chimerism after delivery to air-liquid interface cultures stimulated to produce IL8 by an inflammatory challenge. Finally, engraftment efficiency will be assessed in vivo by delivering engineered airway cells to the murine trachea and by tracking real-time cell migration by two-photon microscopy. Engineering cells to home to the stem cell niche with minimal injury requirements would be a monumental step toward increasing the safety and efficacy of CF cell therapy. The proof-of-concept studies proposed here will bring us one step closer to this goal.

抽象/项目总结