GENE TRANSFER TO AIRWAY EPITHELIAL CELLS

基因转移至气道上皮细胞

• 批准号: 2501436
• 负责人: PI-WAN CHENG
• 金额: $ 18.55万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2501436
• 关键词: chloride channels; cystic fibrosis; gene therapy; human tissue; laboratory mouse; liposomes; method development; respiratory epithelium; tissue /cell culture; transfection /expression vector

DESCRIPTION (Applicant's abstract): Cystic Fibrosis (CF) is the most common lethal genetic disease among Caucasians. Lung diseases account for greater than 95 percent of the morbidity and mortality. Hence, CF lungs have been targeted for gene therapy. Current gene therapy vectors suffer from low transfection efficiency or induction of host immune response, which preclude them from being used for gene therapy in vivo and invite development of alternative vectors. We have developed a novel gene transfer vector composed of a receptor ligand and a cationic liposome, which yields high transfection efficiency in HeLa cells and immortalized tracheal epithelial cells of a cystic fibrosis patient (CFT1). The formation of liposome-transferrin-DNA complexes correlates with high transfection efficiency. The transfection vectors which contain transferrin, insulin, or cholera toxin could correct the cAMP-dependent chloride conductance defect in CFT1 cells. We propose to test the hypothesis that the liposome-receptor ligand-DNA complex by Sepharose gel chromatography and then characterize the physicochemical properties of the putative transfection complex by biochemical and transmission electron microscopic methods. We will examine if this complex alone or in combination with the receptor ligand and/or liposome yields high transfection efficiency in CFT1 cells, primary cultures of mouse and human airway epithelial cells, respiratory epithelial explants, and then in mouse airway epithelia in vivo. We will also characterize the kinetics of the receptor ligand-facilitated gene transfer using confocal microscopic, biochemical, molecular biological, and immunological techniques to identify the step(s) responsible for the enhancement of the transfection efficiency of liposome-mediated gene transfer. The efficacy of the gene therapy protocols employing a plasmid containing the cDNA encoding wild-type cystic fibrosis transmembrane conductance regulator will be examined in the primary cultures of airway epithelial cells and nasal and tracheal epithelia of CF mouse in vivo. The gene transfer vectors will be assessed for inflammatory and immune responses and cytopathology in normal and CF mouse. Correction of cAMP-dependent chloride conductance defect coupled with no immune response to the vectors in CF mouse should be the crucial information needed for planning future human gene therapy trials.

描述（申请人的摘要）：囊性纤维化（CF）是最常见的 高加索人的致命遗传疾病。 肺部疾病较大 超过95％的发病率和死亡率。 因此，CF肺一直 针对基因治疗。 当前的基因疗法载体患有低 转染效率或宿主免疫反应的诱导，这排除了 它们是在体内用于基因疗法的，并邀请开发 替代媒介。 我们已经开发了一种新型的基因转移载体 由受体配体和阳离子脂质体组成，产生高 HeLa细胞的转染效率和永生的气管上皮 囊性纤维化患者的细胞（CFT1）。 形成 脂质体转化蛋白-DNA复合物与高转染相关 效率。 包含转铁蛋白，胰岛素或 霍乱毒素可以纠正依赖CAMP的氯电导缺陷 在CFT1细胞中。 我们建议检验脂质体受体的假设 Sepharose凝胶色谱法配体-DNA复合物，然后表征 假定转染复合物的理化特性 生化和透射电子显微镜方法。 我们将检查 如果单独或与受体配体和/或结合使用该复合物 脂质体在CFT1细胞中产生高转染效率，原代培养物 小鼠和人类气道上皮细胞，呼吸性上皮外植体， 然后在体内的小鼠气道上皮。 我们还将表征 使用共共聚焦的受体配体辅助基因转移的动力学 微观，生化，分子生物学和免疫学技术 确定负责增强转染的步骤 脂质体介导的基因转移的效率。 基因的功效 使用含有编码野生型cDNA的质粒的治疗方案 囊性纤维化跨膜电导调节剂将在 气道上皮细胞以及鼻和气管上皮的原发性培养物 cf鼠标在体内。 将评估基因转移向量 正常和CF小鼠的炎症和免疫反应以及细胞病理学。 校正依赖营地的氯导率缺陷与NO CF小鼠中对向量的免疫反应应为关键信息 计划未来的人类基因治疗试验所需。