A system for the propagation of adenoviral vectors with genetically modified receptor specificities

The development of genetically modified adenovirus (Ad) vectors with specif icity for a single cell type will require both the introduction of novel tr opism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the p ropagation of these targeted Ad vectors. Based on the concept that Ad enter s cells by a two-step process in which a primary receptor serves as a high affinity binding site for the Ad fiber knob, with subsequent internalizatio n mediated by av integrins, we designed two artificial primary receptors. T he extracellular domain of one of these synthetic receptors was derived fro m a single-chain antibody (sFv) with specificity for Ad5 knob, while the se cond receptor consisted of an icosapeptide identified by biopanning a phage display library against Ad5 knob. Expression of either of these artificial virus-binding receptors in fiber receptor-negative cells possessing cry in tegrins conferred susceptibility to Ad infection. We then created a novel m echanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporat ed at the C-terminal of the Ad fiber protein. The resultant Ad vector was a ble to infect nonpermissive cells displaying the cognate artificial recepto r, containing an anti-His sFv. This strategy, comprising a genetically engi neered Ad virion and a modified cell line, should be useful in the propagat ion of targeted Ad vectors that lack the ability to bind the native fiber r eceptor.