GENE TRANSFER TO AIRWAY EPITHELIAL CELLS

基因转移至气道上皮细胞

• 批准号: 2857877
• 负责人: PI-WAN CHENG
• 金额: $ 19.46万
• 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/2857877
• 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