GENE TRANSFER TO AIRWAY EPITHELIAL CELLS

描述(申请人摘要)：囊性纤维化是最常见的 高加索人中的致命遗传病。肺部疾病所占比例更大 超过95%的发病率和死亡率。因此，慢性阻塞性肺疾病的肺一直 以基因治疗为目标。目前的基因治疗载体存在低 转染率或诱导宿主免疫反应，这排除了 它们被用于体内的基因治疗，并邀请开发 可选的载体。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，产率高 HeLa细胞和永生化气管上皮细胞的转染率 囊性纤维化患者的细胞(CFT1)。形成了一种 脂质体-转铁蛋白-DNA复合体与高转染率的相关性 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CFT1细胞中。我们建议检验这样的假设：脂质体-受体 通过琼脂糖凝胶层析得到配体-DNA复合体，并对其进行表征 可能的转导复合体的理化性质 生化和透射电子显微镜方法。我们将研究 如果该复合体单独或与受体配体和/或 脂质体在原代培养CFT1细胞中的高效转染率 小鼠和人类呼吸道上皮细胞，呼吸道上皮外植体， 然后在体内的小鼠呼吸道上皮细胞中。我们还将描述 用共聚焦技术研究受体配基促进基因转移的动力学 显微、生化、分子生物学和免疫学技术 确定促进转染率的步骤(S) 脂质体介导的基因转移的效率。该基因的功效 使用含有编码野生型的cDNA质粒的治疗方案 囊性纤维化跨膜电导调节剂将在 呼吸道上皮细胞和鼻、气管上皮的原代培养 在活体条件下对CF小鼠的免疫抑制作用。基因转移载体将被评估为 正常和CF小鼠的炎症和免疫反应及细胞病理学。 CAMP依赖的氯离子电导缺陷与NO偶联的校正 CF小鼠对载体的免疫应答应是关键信息 计划未来的人类基因治疗试验所需的。

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.