A system for the propagation of adenoviral vectors with genetically modified receptor specificities.
The development of genetically modified adenovirus (Ad) vectors with specificity for a single cell type will require both the introduction of novel tropism determinants and the ablation of endogenous tropism. Consequently, it will not be possible to exploit the native cellular entry pathway in the propagation of these targeted Ad vectors. Based on the concept that Ad enters 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 internalization mediated by alpha v integrins, we designed two artificial primary receptors. The extracellular domain of one of these synthetic receptors was derived from a single-chain antibody (sFv) with specificity for Ad5 knob, while the second 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 alpha v integrins conferred susceptibility to Ad infection. We then created a novel mechanism for cell binding by genetically modifying both the vector and the target cell. In this approach, six histidine (His) residues were incorporated at the C-terminal of the Ad fiber protein. The resultant Ad vector was able to infect nonpermissive cells displaying the cognate artificial receptor, containing an anti-His sFv. This strategy, comprising a genetically engineered Ad virion and a modified cell line, should be useful in the propagation of targeted Ad vectors that lack the ability to bind the native fiber receptor.
Douglas
J T
JT
Gene Therapy Center, Department of Medicine, The University of Alabama at Birmingham, 35294, USA.
Miller
C R
CR
Kim
M
M
Dmitriev
I
I
Mikheeva
G
G
Krasnykh
V
V
Curiel
D T
DT
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D002213:Capsid / Q000235:genetics* / Q000378:metabolism; D036022:Capsid Proteins; D002460:Cell Line; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D005822:Genetic Vectors; D005910:Glioma; D006367:HeLa Cells; D006801:Humans; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014771:Virion / Q000235:genetics
D036022:Capsid Proteins; D011991:Receptors, Virus; C018315:hexon capsid protein, Adenovirus