STOICHIOMETRY OF RECOMBINANT CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN EPITHELIAL-CELLS AND ITS FUNCTIONAL RECONSTITUTION INTOCELLS IN-VITRO

We have generated several clones of Chinese hamster ovary, mouse epith eloid C127, and pig kidney epithelial LLCPK1 cells producing high leve ls of functional recombinant human cystic fibrosis transmembrane condu ctance regulator (CFTR). Processing of CFTR to the mature and fully gl ycosylated form in these cells is inefficient with only approximately 40% of all newly synthesized CFTR being converted to the mature form. Furthermore, expression of the most frequent mutant allele of the cyst ic fibrosis (CF) gene, the DELTAF508 mutant in these epithelial cells, indicated that it is biosynthetically arrested at the endoplasmic ret iculum and fails to traffic to the plasma membrane. Using a combinatio n of CFTR mutants and monoclonal antibodies, all the detectable recomb inant CFTR in these cells was determined at least under the conditions used, to be present as a monomer. To demonstrate the feasibility of p rotein replacement therapy, we were able to effect the physical transf er of functional recombinant CFTR produced in Chinese hamster ovary ce lls to the plasma membranes of Ha3b fibroblasts, a cell line devoid of cAMP-stimulated chloride channels. Transfer of CFTR was mediated by t he hemagglutinin viral fusion protein of influenza virus present on th e Ha3b cells. Efficiency of transfer was up to 25% of the target cells , and CFTR chloride channel activity was detectable for up to 12 h pos t-fusion. Therefore, with the development of an appropriate formulatio n of fusogenic proteoliposome or virosome containing reconstituted pur ified CFTR, it should be feasible to introduce functional CFTR into CF -affected cells.