RECEPTOR-MEDIATED GENE-TRANSFER TO AIRWAY EPITHELIAL-CELLS IN PRIMARYCULTURE

A variety of methods have been utilized for gene transfer to the cells of the airway epithelium. These have included DNA-mediated mechanisms of gene transfer as well as recombinant viral vectors. Despite the av ailability of these methods, limitations in their utility warrant the development of alternate systems. As an alternative, receptor-mediated endocytosis using transferrin-polylysine conjugates has been shown to transduce immortalized airway epithelial cells efficiently via a phys iologic pathway. When transferrin-polylysine conjugates were used to t ransduce airway epithelial cells grown in primary culture, however, ge ne transfer occurred inefficiently. Investigation into this relative i nefficiency centered on endosomal entrapment of the conjugate-DNA comp lex. Pretreatment of the cells with chloroquine, which causes vacuoliz ation and disruption of the endosome, or co-delivery of adenoviral par ticles, which serves to lyse the endosomal membrane, were both associa ted with greatly improved gene transfer efficiency. These studies esta blished that the relative refractory state of the airway epithelial ce lls in primary culture was secondary to the retention of the internali zed material within the endosome. We thus explored the efficiency of c onjugates that possessed a mechanism to escape this endosomal entrapme nt; adenovirus-polylysine conjugates and transferrin-polylysine/adenov irus-polylysine conjugates were thus employed. Gene transfer efficienc y improved significantly with the adenovirus-containing conjugates. Th ese data support the concept that conjugates can be synthesized that m ediate highly efficient gene transfer to airway epithelial cells in pr imary culture via the receptor-mediated endocytosis pathway.