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Mechanism of Restriction of Normal and Cystic Fibrosis Transmembrane Conductance Regulator–Deficient Human Tracheal Gland Cells to Adenovirus Infection and Ad-Mediated Gene Transfer

Laboratoire de Virologie et Pathogénèse Virale, Faculté de Médecine RTH Laennec, Lyon; Transgene SA, Strasbourg; and Groupe de Recherche sur les Glandes Endocrines, Faculté de Médecine, Marseille, France

Address correspondence to: Pr. Pierre Boulanger, Laboratoire de Virologie et Pathogénèse Virale, Faculté de Médecine RTH Laennec de Lyon, 7, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France. E-mail: Pierre.Boulanger{at}laennec.univ-lyon1.fr

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)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 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 [26]-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 Ad5 virions. Ad5 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, Ad5 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 Ad5 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 Ad5 vectors carrying fibers terminated by their natural knob domain.

Abbreviations: adenovirus, Ad • Coxsackie-Ad receptor, CAR • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • Dulbecco's modified Eagle's medium, DMEM • electron microscopy, EM • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • fast performance liquid chromatography, FPLC • heparan sulfate glycosaminoglycans, HS-GAG • multiplicity of infection, MOI • phosphate-buffered saline, PBS • plaque-forming units, PFU • post-infection, pi • Arg-Gly-Asp, RGD • sialic acid, SA • sialoglycoproteins, SAGP • sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE • wild-type, WT

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