Research Project
3366322
DESCRIPTION (adapted from the application): The proposed work will make use of newly gained information concerning the gene responsible for the defect in cystic fibrosis (CF) to develop approaches to use the cystic fibrosis transmembrane conductance regulator (CFTR) as a direct protein replacement therapy for treatment of the disease. The eventual goal is the development of an aerosol containing a fusogenic, targeted preparation of purified CFTR in a lipid-containing formulation capable of directly delivering protein to defective cells of the airway epithelia in CF patients. The cDNA for CFTR has been isolated and cloned into a number of mammalian cell expression vectors including those with inducible promoters. DNA will be transfected into C127, Chinese hamster ovary (CHO) cells, and cells capable of apocrine secretion. Cells expressing reasonable levels of protein will be isolated. Polyclonal and monoclonal antibodies have been raised to fusion proteins containing sequences from CFTR. Plasma membranes containing CFTR from non-recombinant and recombinant cells and membrane vesicles or blebs resulting from apocrine secretion from recombinant cells will be prepared. These will be fused chemically to nasal epithelial cells from CF patients and changes in ion efflux measured. The ability to correct the CF phenotype in tissue culture cells will indicate production, isolation, and delivery of active CFTR. This will be followed by experiments to purify the CFTR protein and to reconstitute it into lipid containing-vesicles. In parallel, attempts will be made to increase the efficiency and specificity of fusing the CFTR protein into cells. The approach will be to use the hemagglutinin and fusion glycoproteins of viruses with tropism for airway epithelial cells. Plasma membrane preparations, blebs, virosomes, and liposomes all containing viral glycoproteins and CFTR will be used to target to CF airway cells and ion efflux measured. Following successful transfer of CFTR into cells, the incorporated protein will be characterized immunologically and electrophysiologically in addition to being tested in the efflux assay. Conditions for growth of recombinant cells in large scale production will be established and the purification process developed to at least the 10 mg scale. Successful completion of the experiments will allow initiation of animal experiments and refinement of the CFTR protein delivery system.
