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.
| Baatz, J E; Zou, Y; Korfhagen, T R (2001) Inhibitory effects of tumor necrosis factor-alpha on cationic lipid-mediated gene delivery to airway cells in vitro. Biochim Biophys Acta 1535:100-9 |
| Ross, G F; Bruno, M D; Uyeda, M et al. (1998) Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. Gene Ther 5:1244-50 |
