TARGETED TRANSFECTION OF THE PULMONARY EPITHELIUM

正如本提案所概述的，我们将检验我们的假设，即基因 由肺细胞特异性启动子控制的细胞因子可以被递送到肺细胞。 气道上皮表面活性蛋白A（SP-A）和B（SP-B） 复合物作为运载工具。 这些蛋白质是 肺表面活性剂，并且不知道是有毒的或免疫原性的。 这 提案分为三个目标。在Aim I中，我们将开发一个 将DNA递送至肺腺癌细胞的有效方法， H441，在体外利用SP-A和SP-B作为递送的组分 车辆. 天然SP-B的构象提供了阳离子表面， DNA将直接复合。该复合物将在 不存在和存在阳离子脂质和去污剂， DNA转染。 为了提高这些蛋白质的溶解度 增强与DNA的络合作用，SP-A和SP-B将共价结合到 通过添加聚赖氨酸聚合物在N-末端修饰。 以来 这些天然存在的多聚赖氨酸缀合物 蛋白质可能根据其各自的生理特性，我们将 通过傅立叶变换检测修饰蛋白质的结构和功能 变换红外光谱法、表面测定法和 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 优选用于转染测定。 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 目的：观察成年小鼠肺上皮细胞在体内的变化， 经气管注射 将确定转染效率 通过CAT测定。 将使用最有效的转染方法 通过原位杂交进行细胞内表达定位， CAT mRNA。 原位杂交也将用于确定 表达转染基因的气道上皮细胞的比例 DNA. RSV CAT质粒将用于检查细胞 表达的分布，而SP-C CAT和CC 10 CAT将是 用于靶向肺上皮细胞。 有效的方法 然后将在该目标内建立的细胞用于转染SP-1。 C/CFTR和CCl 0/CFTR靶向CFTR表达至肺 上皮 在Aim III中，我们将采用成功的体内转染 方法来确定的蛋白质的细胞内路由的 递送载体和转染的DNA。 这些研究将是 用于改善向体内气道上皮的递送。知识 从这项拟议的研究中获得的将被应用于转染 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染。

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.