CATIONIC LIPOSOME MEDIATED GENE THERAPY--CYSTIC FIBROSIS

阳离子脂质体介导的基因治疗--囊性纤维化

• 批准号: 2145144
• 负责人: ROBERT J DEBS
• 金额: $ 9.91万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145144
• 关键词: aerosols; chloramphenicol acetyltransferase; chloride channels; cystic fibrosis; drug administration rate /duration; gene expression; gene therapy; genetic enhancer element; genetic promoter element; histology; intravenous administration; laboratory mouse; liposomes; lung; reporter genes; respiratory epithelium; tissue /cell culture; transfection

Gene therapy, the correction of genetic defects by site-specific transgene expression in vivo, will revolutionize the treatment of human disease. The cystic fibrosis transmembrane conductance regulator (CFTR) gene has been clones and specific CFTR mutations which result in cystic fibrosis (CF) have been characterized in detail. However, the inability to achieve extended, high level expression of transgenes in the lung has precluded translation of this molecular cloning achievement into effective gene therapy for CF. Recently, we have demonstrated high levels of chloramphenicol acetyltransferase (CAT) reporter gene expression in the lungs of mice for up to 3 weeks following either aerosolization or intravenous (iv) injection of CAT expression vectors complexed to cationic liposomes. Immunostaining revealed that the majority of airway epithelial cells strongly express the CAT gene in vivo. We observed no histologic evidence of toxicity in any treated mice. We now propose to further develop our in vivo gene delivery technology, in order to create effective gene therapy for CF. Our 3 main goals are: 1) To maximize the level, duration and cellular-specificity of CAT reporter gene expression in the lungs of mice, using cationic liposome- mediated gene delivery by aerosol or iv administration. To accomplish these goals, we will first determine the parameters which maximize cationic liposome-mediated transfection of selected cultured cells. We will then use this data to maximize CA gene expression in mice by optimizing: A) the heterologous promoter element, B) cationic liposome formulation, C) DNA to liposome ratio, D) gene dosage and E) mean aerosol particle size and F) frequency/duration of administration. We will use immunostaining for intracellular CAT protein to identify the specific lung cell types and percentage of these cells transfected in vivo. 2) Maximize the level of duration of CFTR transgene expression in the lung using heterologous promoter elements. We will use the optimized parameters determined for the CAT reporter gene, including the most active viral promoter elements tested, to ten maximize CFTR transgene expression in the lung. 3) Use the CFTR 5' regulatory region to target CFTR transgene expression to appropriate lung cell types in vivo. We will use selected regulatory sequences derived from the 5' CFTR untranslated region fused to the CFTR coding region to mimic the endogenous pattern of CFTR gene expression in normal individuals. In summary, we will optimize the level, duration and cellular specificity of cationic liposome-mediated CFTR transgene expression we can achieve in vivo, while minimizing host toxicity. Our goal is to develop safe and effective, genetically-based in vivo therapy for CF. The ability to express the CFTR transgene in airway epithelial and sub- mucosal cells in vivo is an essential step towards achieving this goal.

基因疗法，按特定地点对遗传缺陷的纠正 转基因在体内的表达将彻底改变人类的治疗 疾病。囊性纤维化跨膜电导调节剂（CFTR） 基因是克隆和特定的CFTR突变，导致囊性 纤维化（CF）已详细表征。但是，无能 为了达到扩展，肺中转基因的高水平表达 排除该分子克隆成就的翻译 有效的CF基因疗法。最近，我们已经证明了很高 氯霉素乙酰转移酶（CAT）报告基因的水平 两者后，在小鼠的肺中表达长达3周 CAT表达载体的雾化或静脉注射（IV）注射 复合到阳离子脂质体。免疫染色表明 大多数气道上皮细胞强烈表达CAT基因 体内。我们观察到任何治疗的毒性的组织学证据 老鼠。我们现在建议进一步发展我们的体内基因递送 技术，为了创建有效的CF基因疗法。我们的3个主 目标是： 1）最大化CAT的水平，持续时间和细胞特异性 使用阳离子脂质体 - 在小鼠肺中的报告基因表达 气溶胶或IV施用介导的基因递送。完成 这些目标，我们将首先确定最大化的参数 阳离子脂质体介导的选定培养细胞转染。我们 然后将使用此数据来最大化小鼠中Ca基因表达 优化：a）异源启动子元素，b）阳离子脂质体 配方，c）DNA与脂质体比率，d）基因剂量和e）平均值 气溶胶粒径和f）给药频率/持续时间。我们 将使用免疫染色进行细胞内CAT蛋白来鉴定 这些转染的这些细胞的特定肺细胞类型和百分比 体内。 2）最大化CFTR转基因表达持续时间 肺使用异源启动子元素。我们将使用优化 针对CAT报告基因确定的参数，包括最多的参数 测试的主动病毒启动子元素，达到十个最大CFTR转基因 在肺中的表达。 3）使用CFTR 5'调节区域靶向CFTR转基因表达 在体内适当的肺细胞类型。我们将使用选定的监管 从融合到CFTR的5'CFTR未翻译区域得出的序列 编码区以模仿CFTR基因表达的内源性模式 正常人。 总而言之，我们将优化水平，持续时间和细胞 阳离子脂质体介导的CFTR转基因表达的特异性 可以实现体内，同时最大程度地减少宿主毒性。我们的目标是 为CF开发安全有效的，基于遗传的体内疗法。 在气道上皮和亚 - 表达CFTR转基因的能力 体内粘膜细胞是实现这一目标的重要一步。