Delivery of genes into the airways by installation or by aerosol permits the luminal delivery of genes. These methods are limited because (i) in diseases such as cystic fibrosis, aerosols will deposit irregularly; (ii) after deposition, the presence of mucus and inflammation limit the ability of the delivered material to interact with airway cells; (iii) cells such as gland serous cells (believed to be important targets for CFTR) are not easily accessible from the airway lumen. For these reasons, we propose to take advantage of three strategies for delivering genes into tissue via the circulation. These strategies include (1) use of liposomes that survive for long periods of time in the circulation; (2) methods for increasing the postcapillary venular permeability to liposomes transiently, so the liposome-DNA complexes can be concentrated in airway tissue; and (3) methods for introducing the DNA into selected cells (e.g. in collaboration with Dr. Carol Basbaum, to use airway submucosal gland serous cell promoters). We have already demonstrated the feasibility of concentrating liposomes selectively in airways. In the proposed studies, we will utilize these newly developed methods to introduce genes into selected cells. We propose to extend the above observations to DNA-carrying sterically stabilized liposomes for a more efficient uptake and expression of the human CFTR gene by specific cells within the bronchial airways. Our proposed project will include: steric stabilization of the DNA-liposome complexes for increasing their circulation time in blood, and: temporary increase of the permeability of the airway endothelial layer to the liposome-DNA complexes by the proper administration of specific agents that allow liposomes to move across the postcapillary venular gaps. The localization of the liposome-DNA complexes will be verified and quantitated biochemically and morphologically, by fluorescence and electron microscopy using techniques that we have recently developed. The presence of the gene product within the recipient cells will be calculated, using morphological and cytochemical methods to identify the transfected cells, and to quantitate the expression of the gene. The work will proceed in the following sequence, first liposome markers followed by marker genes (CAT) and finally constructs containing the human CFTR gene.
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