TARGETED TRANSFECTION OF THE PULMONARY EPITHELIUM

肺上皮的靶向转染

• 批准号: 2145195
• 负责人: Thomas R Korfhagen
• 金额: $ 15.78万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 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 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2145195
• 关键词: chemical conjugate; chloramphenicol acetyltransferase; chloride channels; cystic fibrosis; electron microscopy; gene therapy; in situ hybridization; infrared spectrometry; laboratory mouse; lung; polylysine; protein structure function; pulmonary surfactants; reporter genes; respiratory epithelium; tissue /cell culture; transfection

As outlined in this proposal, we will test our hypothesis that genes controlled by pulmonary cell specific promoters can be delivered to the airway epithelium with surfactant proteins A (SP-A) and B (SP-B) complexes as delivery vehicles. These proteins are natural components of pulmonary surfactant and are not known to be toxic or immunogenic. This proposal is organized into three aims. In Aim I we will develop an efficient method of DNA delivery to the pulmonary adenocarcinoma cells, H441, in vitro utilizing SP-A and SP-B as components of the delivery vehicles. The conformation of native SP-B provides a cationic surface to which DNA will directly complex. This complex will be formulated in the absence and presence of cationic lipid and detergents for optimization of DNA transfection. In order to improve the solubility of these proteins and enhance complexation with DNA, SP-A and SP-B will be covalently modified at the N-terminus by the addition of polylysine polymers. Since it is possible that polylysine conjugation of these naturally occurring proteins may after their respective physiological properties, we will examine the structure and function of modified proteins by Fourier transform-infrared spectroscopy, surfactometry and measurement of inhibition of phospholipid secretion by Type II cells. The modified forms of proteins with minimal effects on these properties will be preferentially utilized in transfection assays. We will quantitate transfection efficiency by CAT assay with the RSV CAT reporter plasmid. In Aim II, we will use the most efficient delivery method determined in Aim I and transfect the pulmonary epithelium of adult mice in vivo by transtracheal injection. Efficiency of transfection will be determined by CAT assay. The most efficient transfection methods will be utilized for intracellular localization of expression by in situ hybridization of CAT mRNA. In situ hybridization will also be used to determine the proportion of airway epithelial cells that are expressing transfected DNA. The RSV CAT plasmid will be used to examine the cellular distribution of expression, whereas SP-C CAT and CC10 CAT will be utilized to target the pulmonary epithelial cells. Efficient methods established within this Aim will then be utilized for transfection of SP- C/CFTR and CCl0/CFTR to target CFTR expression to the pulmonary epithelium. In Aim III we will employ successful in vivo transfection methods to identity the intracellular routing of the protein of the delivery vehicle and the transfected DNA. These latter studies will be utilized to improve delivery to the airway epithelium in vivo. Knowledge gained from this proposed research will be applied to transfection of mouse lungs in models of Cystic Fibrosis, with a long term goal of transfection of primate lungs in vivo.

如本提案中概述的那样，我们将测试我们的假设 由肺细胞特异性启动子控制 带有表面活性剂蛋白A（SP-A）和B（SP-B）的气道上皮 复合物作为送货车辆。 这些蛋白质是天然成分 肺表面活性剂，并非有毒或免疫原性。 这 提案分为三个目标。在目的中，我将开发一个 有效的DNA传递到肺腺癌细胞的方法， H441，在体外利用SP-A和SP-B作为输送的组成部分 车辆。 天然SP-B的构象提供了阳离子表面 哪个DNA将直接复杂。这个综合体将在 阳离子脂质和洗涤剂的不存在和存在以优化 DNA转染。 为了提高这些蛋白质的溶解度 并增强与DNA，SP-A和SP-B的络合 通过添加聚赖氨酸聚合物在N末端修饰。 自从 这些天然发生的聚赖氨酸结合可能 蛋白质可能在各自的生理特性之后，我们将 通过傅立叶检查改性蛋白的结构和功能 转换红外光谱，表面测量法和测量 通过II型细胞抑制磷脂分泌。 修改后 对这些特性的影响最小的蛋白质形式将是 优先用于转染测定法。 我们将定量 用RSV CAT报告质粒通过CAT测定的转染效率。 在AIM II中，我们将使用确定的最有效的交付方法 AIM I并通过体内转染成年小鼠的肺上皮 经口径注射。 将确定转染效率 通过猫测定。 最有效的转染方法将被使用 用于通过原位杂交表达的细胞内定位 猫mrna。 原位杂交也将用​​于确定 表达转染的气道上皮细胞的比例 脱氧核糖核酸。 RSV CAT质粒将用于检查细胞 表达的分布，而SP-C CAT和CC10 CAT将是 用于靶向肺上皮细胞。 有效的方法 然后，在此目标中建立的将用于转染Sp- C/CFTR和CCL0/CFTR以靶向CFTR表达到肺部 上皮。 在AIM III中，我们将在体内转染中成功使用 识别蛋白质内蛋白质内路线的方法 输送车辆和转染的DNA。 这些后一个研究将是 用于改善体内气道上皮的递送。知识 从这项拟议的研究中获得的将应用于转染 囊性纤维化模型中的小鼠肺，长期目标的 在体内转染灵长类动物肺。