TARGETED TRANSFECTION OF THE PULMONARY EPITHELIUM

正如这项提案中所概述的，我们将检验我们的假设 由肺细胞特异性启动子控制的启动子可以传递到 含表面活性蛋白A(SP-A)和B(SP-B)的呼吸道上皮细胞 作为运输工具的建筑群。这些蛋白质是天然成分 肺表面活性物质，目前尚不清楚其毒性或免疫原性。这 提案分为三个目标。在AIM I中，我们将开发一种 向肺腺癌细胞输送DNA的有效方法， H441，体外利用SP-A和SP-B作为递送成分 车辆。天然SP-B的构象提供了阳离子表面 其中的DNA会直接复杂化。这个综合体将在 阳离子脂类和洗涤剂的存在和不存在，用于优化 DNA转基因技术。为了提高这些蛋白质的溶解度 并增强与DNA的络合作用，SP-A和SP-B将共价 通过添加多聚赖氨酸聚合物在N-末端进行修饰。自.以来 这些多聚赖氨酸天然结合是可能的 蛋白质可能会根据它们各自的生理特性，我们会 傅立叶变换法检测修饰蛋白质的结构和功能 变换红外光谱分析、表面光谱分析和测量 抑制II型细胞的磷脂分泌。修改后的 对这些性质影响最小的蛋白质形式将是 在转染法中优先使用。我们将量化 用RSV CAT报告质粒进行CAT检测，结果表明，RSV CAT报告质粒具有较高的转染率。 在AIM II中，我们将使用下列确定的最有效的交付方法 目的1.通过基因工程技术对成年小鼠肺上皮细胞进行体内转基因研究。 经气管注射。转染率将被确定 过氧化氢酶测定法。将利用最有效的转染法 通过原位杂交进行细胞内表达的定位 CAT基因的表达。还将使用原位杂交来确定 表达转基因的呼吸道上皮细胞的比例 DNARSV CAT质粒将被用来检测细胞 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 在这一目标下建立的将被用于SP-1的转染 C/cftr和ccl0/cftr靶向肺组织cftr表达 上皮组织。在AIM III中，我们将采用成功的体内转染法 鉴定蛋白质在细胞内的路径的方法 运送工具和转染体DNA。这些后面的研究将是 用于改善体内对呼吸道上皮的输送。知识 从这项拟议的研究中获得的结果将被应用于转基因 囊性纤维化模型中的小鼠肺，长期目标是 灵长类动物肺的体内转基因。

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.