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

Abstract

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 alpha[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 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.

Enhancement of Adenovirus-Mediated Gene Delivery to Rheumatoid Arthritis Synoviocytes and Synovium by Fiber Modifications: Role of Arginine-Glycine-Aspartic Acid (RGD)- and Non-RGD-Binding Integrins

“…Pb, R340E and D288K mutant, hexon or fiber knob), as described previously (22,23). Briefly, 1 ϫ 10 5 RA FLS were incubated with hexon (3, 30, or 300 ng total protein), Pb protein, WT or mutant (0.5, 5, or 50 ng), or knob protein (4, 40 or 400 ng) for 1 h at 4°C, in 200 l/well of a 24-well plate.…”

“…The knob domain of Ad5 is known to bind other attachment receptors apart from CAR, which includes heparin sulfate glycosaminoglycans and the MHC class I ␣2 domain (23,43,44). We investigated the role of the knob domain in Ad5GFP-R7-knob entry of FLS by cell binding competition assays.…”

The Maturation of Murine Dendritic Cells Induced by Human Adenovirus Is Mediated by the Fiber Knob Domain

“…However, the partial overlapping of the DC-binding site(s) on the knob with those specific for CAR is not coincidental, if one considers that the regions of the knob involved in the interaction with cell components must be very exposed on the protein surface and thus accessible to many other receptors and/or ligands. For example, a CAR-blocking anti-knob monoclonal antibody has been found to inhibit the binding of Ad5 virions to alternative receptors, heparan sulfate proteoglycans and sialoglycoproteins (37).…”

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…As the FibDCAR-HI-Link-ZHZH fiber does not bind to CAR, it would be reasonable to suggest that the anti-knob antibody effect in this case was caused by a steric hindrance, as has been reported for alternative Ad5 receptors before. 43 Taken together, these results suggest that the recombinant fibers were specific with respect to their inserted Her2 Affibody, and that deletion of CAR and the insertion of the Affibody molecule ZH did not introduce any unwantedbinding properties.…”