GENE-TRANSFER TO RESPIRATORY EPITHELIAL-CELLS VIA THE RECEPTOR-MEDIATED ENDOCYTOSIS PATHWAY

Gene-based therapies for a variety of inherited and acquired pulmonary diseases will require the development of vectors capable of safe and efficient transfer of DNA to the respiratory epithelium. The present study examined the feasibility of delivering DNA to respiratory epithelial cells by the receptor-mediated endocytosis pathway. This strategy employs molecular conjugates consisting of a cognate moiety, in this case human transferrin, covalently linked to a DNA-binding moiety, such as a cationic polyamine. Complexes were formed between transferrin-polylysine conjugates (hTfpL) and plasmid DNA carrying the firefly luciferase reporter gene (pRSVL). The conjugate-DNA complexes were added directly to cells in tissue culture and incubated for 24 h, after which cell lysates were analyzed for luciferase enzyme activity by luminometry. An immortalized human respiratory epithelial cell line (HBE1) treated with the transferrin-polylysine-DNA complexes exhibited luciferase enzyme activity significantly augmented over background levels. This respiratory epithelial cell line exhibited greater susceptibility to gene transfer by the transferrin-polylysine conjugates than did non-respiratory epithelial cell lines known to possess high levels of transferrin receptors. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the conjugates by the transferrin pathway was verified by competition for the transferrin receptor. In addition, treatment with agents that either increased transferrin receptor number or decreased lysosomal degradation markedly augmented gene expression mediated by the conjugates. Thus, respiratory epithelial cells possess receptors for transferrin that can be exploited to accomplish gene transfer by the receptor-mediated endocytosis pathway.