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（申请人摘要）：囊性纤维化（CF）是最常见的 白种人中致命的遗传病。 肺部疾病占较大比例 95%以上的发病率和死亡率。 因此，CF肺已 基因治疗的目标。 目前的基因治疗载体存在低 转染效率或宿主免疫反应的诱导，这排除了 它们被用于体内基因治疗并邀请开发 替代向量。 我们开发了一种新型基因转移载体 由受体配体和阳离子脂质体组成，产率高 HeLa细胞和永生化气管上皮细胞的转染效率 囊性纤维化患者的细胞（CFT1）。 的形成 脂质体-转铁蛋白-DNA 复合物与高转染相关 效率。 转染载体含有转铁蛋白、胰岛素或 霍乱毒素可以纠正 cAMP 依赖性氯电导缺陷 在 CFT1 细胞中。 我们建议检验脂质体受体的假设 配体-DNA 复合物通过琼脂糖凝胶层析，然后表征 推定转染复合物的理化特性 生物化学和透射电子显微镜方法。 我们将检查 如果该复合物单独或与受体配体组合和/或 脂质体在 CFT1 细胞、原代培养物中产生高转染效率 小鼠和人类气道上皮细胞、呼吸道上皮外植体、 然后在小鼠体内气道上皮细胞中进行。 我们还将描述 使用共聚焦受体配体促进基因转移的动力学 显微镜、生物化学、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。 基因功效 使用含有编码野生型的cDNA的质粒的治疗方案 囊性纤维化跨膜电导调节器将在 气道上皮细胞以及鼻和气管上皮细胞的原代培养物 CF小鼠体内。 基因转移载体将被评估 正常和 CF 小鼠的炎症和免疫反应以及细胞病理学。 纠正 cAMP 依赖性氯离子电导缺陷以及无 CF小鼠对载体的免疫反应应该是关键信息 规划未来人类基因治疗试验所需。