GENE TRANSFER TO AIRWAY EPITHELIAL CELLS

描述（申请人摘要）：囊性纤维化（CF）是最常见的 致命的遗传病 肺部疾病占 超过95%的发病率和死亡率。 因此，CF肺一直是 用于基因治疗 目前的基因治疗载体具有低的 转染效率或诱导宿主免疫应答，这排除了 它们被用于体内基因治疗，并邀请开发 替代载体。 我们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， HeLa细胞和永生化气管上皮细胞中的转染效率 囊性纤维化患者的细胞（CFT 1）。 的形成 脂质体-转铁蛋白-DNA复合物与高转染相关 效率 含有转铁蛋白、胰岛素或 霍乱毒素可纠正cAMP依赖的氯离子电导缺陷 在CFT 1细胞中。 我们建议测试的假设，脂质体受体 通过琼脂糖凝胶色谱法分析配体-DNA复合物，然后对配体-DNA复合物进行表征。 的物理化学性质的推定的转染复合物， 生物化学和透射电子显微镜方法。 我们将研究 如果该复合物单独或与受体配体和/或 脂质体在CFT 1细胞、原代培养物中产生高转染效率 小鼠和人气道上皮细胞，呼吸道上皮外植体， 然后在小鼠体内气道上皮中。 我们还将描述 受体配体促进的基因转移的动力学 显微镜、生物化学、分子生物学和免疫学技术 鉴定负责增强转染的步骤 脂质体介导的基因转移效率。 基因的功效 使用含有编码野生型的cDNA的质粒的治疗方案 囊性纤维化跨膜传导调节因子将在 气道上皮细胞以及鼻和气管上皮细胞的原代培养物 CF小鼠体内的 将评估基因转移载体的 炎症和免疫反应以及细胞病理学。 cAMP依赖性氯离子电导缺陷与NO偶联的校正 CF小鼠对载体的免疫应答应该是关键信息 用于规划未来的人类基因治疗试验。

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.