Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus infection and ad-mediated gene transfer.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)S infection and Ads-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ads interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that a[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ads virions. Ads attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ads vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ads to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ads vectors carrying fibers terminated by their natural knob domain.