CATIONIC LIPOSOME-BASED GENE THERAPY FOR LUNG CANCER

基于阳离子脂质体的肺癌基因治疗

• 批准号: 3203011
• 负责人: ROBERT J DEBS
• 金额: $ 15.21万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3203011
• 关键词: antineoplastics; cations; cell type; colony stimulating factor; gene dosage; gene expression; gene therapy; genetic enhancer element; genetic promoter element; laboratory mouse; liposomes; lung neoplasms; melanoma; 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. Molecular defects underlying human lung malignancy, including the over expression of transforming oncogenes or the loss of tumor suppressor genes, have been characterized in detail. However, the inability to achieve high level expression of transgenes in the lung has precluded translation of these molecular analyses into effective gene therapy for lung cancer. We have demonstrated high levels of transgene expression in the lungs of mice for up to 60 days following either aerosolization or intravenous (iv) injection of expression vectors complexed to cationic liposomes into normal animals. We have also efficiently transfected metastatic lung tumors and intravascular tumor emboli after iv injection of cationic liposome-reporter gene complexes into tumor-bearing mice. We have documented high level expression of CFTR, GM-CSF and CAT transgenes in the lung, and have observed no histologic evidence of toxicity in any treated mice. We now propose to further develop our in vivo gene delivery technology, to create effective gene-based therapies for human lung cancer. Our 2 main goals are: 1) To maximize the level, duration and cellular-specificity of GM-CSF and p53 transgene expression we can achieve in the lungs, and specifically within lung tumors of tumor-bearing mice by cationic liposome-mediated aerosol and/or iv gene delivery. To accomplish these goals, we will first determine the parameters which maximize cationic liposome-mediated transfection of cultured cells. Second, we will use this data to maximize transgene expression in rodents by optimizing: A) transgene promoter-enhancer element, B) cationic liposome formulation, C) DNA to liposome ratio, D) gene dosage and E) frequency/duration of administration. We will use immunostaining to identify the specific cell types which are transfected in vivo. 2) To then maximize the anti-tumor effects of the p53 and GM-CSF transgenes in tumor-bearing mice. Specifically, we will test whether A) high level, intrapulmonary expression of the GM-CSF transgene can produce significant anti-tumor effects in mice bearing a lung metastatic subclone of B-16 melanoma, or B) expression of the wildtype p53 transgene in vivo can produce anti-tumor effects against the human lung cancer line NCI- H358, grown in the lungs of nude mice. NCI-H358 carries a homozygous deletion of p53, and this line is growth arrested in culture after lipofection of a wild-type p53 expression plasmid. In summary, we will optimize the level, duration and cellular specificity of cationic liposome-mediated transgene expression we can now achieve in vivo, while minimizing host toxicity. Our overall goal is to develop safe and effective, genetically-based in vivo treatments for lung cancer. The ability to express transgenes in the lung, and specifically within lung tumors 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。 人类肺恶性肿瘤的分子缺陷，包括 转化的癌基因或肿瘤丧失的过度表达 抑制基因已详细介绍。 但是， 无法实现肺中转基因的高水平表达 这些分子分析不排除将其转换为有效基因 肺癌治疗。 我们已经证明了高水平的转基因 两只小鼠的肺表达最多60天 表达向量的雾化或静脉注射（IV）注射 将阳离子脂质体复合到正常动物中。 我们也有 有效转染转移性肺肿瘤和血管内肿瘤 静脉注射阳离子脂质体复合蛋白基因复合物后的栓子 进入肿瘤小鼠。 我们已经记录了高水平的表达 CFTR，GM-CSF和肺中的CAT转基因，并且没有观察到 任何治疗小鼠的毒性的组织学证据。 我们现在建议 进一步发展我们的体内基因输送技术，以创造有效 基于基因的人肺癌疗法。 我们的两个主要目标是： 1）最大化GM-CSF的水平，持续时间和细胞特异性和 p53转基因表达我们可以在肺部，特别是 阳离子脂质体介导的肿瘤小鼠肺肿瘤内 气溶胶和/或IV基因递送。 为了实现这些目标，我们将 首先确定最大化阳离子脂质体介导的参数 转染培养细胞。 其次，我们将使用这些数据来 通过优化：a）转基因在啮齿动物中最大化转基因表达 启动子 - 增强剂元素，b）阳离子脂质体配方，c）DNA至 脂质体比率，d）基因剂量和e）频率/持续时间 行政。 我们将使用免疫染色来识别特定细胞 在体内转染的类型。 2）然后最大化p53和GM-CSF的抗肿瘤效应 肿瘤小鼠中的转基因。 具体来说，我们将测试是否a） GM-CSF转基因的高水平，肺内表达可以产生 在带有肺转移亚克隆的小鼠中显着的抗肿瘤作用 B-16黑色素瘤或b）体内野生型p53转基因的表达 可以针对人类肺癌系列产生抗肿瘤作用。 H358，生长在裸鼠的肺中。 NCI-H358携带纯合子 删除p53，这条线是在文化中被捕的 野生型p53表达质粒的脂触。 总而言之，我们将优化水平，持续时间和细胞特异性 阳离子脂质体介导的转基因表达，我们现在可以实现 体内，同时最大程度地减少宿主毒性。 我们的总体目标是发展 安全有效的，基于遗传的肺癌治疗。 在肺中，特别是在内部表达转基因的能力 肺部肿瘤是实现这一目标的重要一步。