RECEPTOR-MEDIATED GENE-TRANSFER TO AIRWAY EPITHELIAL-CELLS IN PRIMARY CULTURE

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 availability 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 physiologic pathway. When transferrin-polylysine conjugates were used to transduce airway epithelial cells grown in primary culture, however, gene transfer occurred inefficiently. Investigation into this relative inefficiency centered on endosomal entrapment of the conjugate-DNA complex. Pretreatment of the cells with chloroquine, which causes vacuolization and disruption of the endosome, or co-delivery of adenoviral particles, which serves to lyse the endosomal membrane, were both associated with greatly improved gene transfer efficiency. These studies established that the relative refractory state of the airway epithelial cells in primary culture was secondary to the retention of the internalized material within the endosome. We thus explored the efficiency of conjugates that possessed a mechanism to escape this endosomal entrapment; adenovirus-polylysine conjugates and transferrin-polylysine/adenovirus-polylysine conjugates were thus employed. Gene transfer efficiency improved significantly with the adenovirus-containing conjugates. These data support the concept that conjugates can be synthesized that mediate highly efficient gene transfer to airway epithelial cells in primary culture via the receptor-mediated endocytosis pathway.