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

• 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
• 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

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.

摘要/项目摘要 囊性纤维化（CF）是一种遗传性疾病，其中囊性纤维化跨膜调节因子（CFTR） 蛋白质缺失或功能失调，导致粘液积聚，粘液纤毛运输受损， 慢性感染和炎症，最终导致死亡。 CF 的病因很难治疗， 300 种已知的致病蛋白质变异体。细胞疗法提供了一种突变盲治疗选择；然而，细胞 植入高阻力气道上皮是无效的，并且需要广泛的气道损伤。 促进细胞穿过紧密连接屏障并将细胞直接输送至干细胞的方法 细胞生态位可能是一种替代方案。尽管细胞治疗模型具有挑战性，但细胞易位 上皮屏障并非没有生物学先例。中性粒细胞很容易通过以下方式迁移到空气空间中： 趋化性，细胞响应化学刺激物的定向迁移。这种运动开始时 中性粒细胞表面的趋化因子受体结合目标趋化因子。在肺部，产生趋化因子 由基底细胞、上呼吸道干细胞和干细胞生态位的主要居民组成。这个过程是 与 CF 尤为相关，因为 CF 中细菌定植是慢性的，并且免疫反应猖獗。 尽管在中性粒细胞和其他免疫细胞中对趋化性进行了充分研究，但尚不清楚是否可以人工表达 非免疫原代细胞中的趋化因子受体将使它们能够通过紧密连接屏障进行趋化。 我们实验室的初步结果表明，原代人支气管上皮细胞 (HBEC) 被设计为 在白细胞介素 8 (IL8) 梯度存在的情况下表达趋化因子受体 CXCR1 趋化性。像这样， 我们的中心假设是，改造 HBEC 来表达趋化因子受体将促进定向 向趋化因子源（即干细胞生态位）迁移，从而提高细胞的效率 植入。为了检验这一假设，将使用不同的慢病毒在 HBEC 中优化 CXCR1 表达 发起人。 CXCR1 丰度将通过蛋白质印迹进行量化，定向迁移将在 二维和三维趋化性测定。典型趋化通路成员将通过以下方式量化 蛋白质印迹和荧光报告基因检测。将外源递送的细胞导向干细胞的目标 细胞生态位最终是为了以最小的损伤要求来增加长期的细胞植入。因此， 工程化 HBEC 的植入效率将通过量化交付后的嵌合状态进行体外评估 气液界面培养物通过炎症刺激刺激产生 IL8。最后，植入 将通过将工程气道细胞递送至小鼠气管并跟踪来评估体内效率 双光子显微镜实时细胞迁移。工程化细胞以最小的成本定位到干细胞生态位 损伤要求将是提高 CF 细胞疗法安全性和有效性的重要一步。 这里提出的概念验证研究将使我们更接近这一目标。