Adenovirus stripping: a versatile method to generate adenovirus vectors with new cell target specificity.
We developed a new type of adenovirus type 5 (Ad5)-derived vector with genetically modified fiber proteins whose knob domains could be stripped off due to the insertion of a single Factor Xa cleavage site in the fiber shaft, between a cellular ligand and the knob domain. This Ad vector did not require a specific cell line for propagation and could be grown in HEK-293 cells. Stripping off the knob domains removed the endogenous cell-binding moiety of Ad but retained the new cell ligand for retargeting purposes. As experimental models for cell ligands, we used two peptides with different sequence complexities: (i) the integrin-binding tripeptide RGD and (ii) a 58-residue oligopeptide termed affibody (Zwt). Zwt binds specifically to the human IgG1 Fc domain or to its Fc3(1) homolog. The modified fibers were efficiently encapsidated into virions, and the Factor Xa sites were fully accessible to proteolysis. In vitro binding assays using recombinant Fc3(1) protein and Ad5-mediated gene transduction of Fc3(1)-expressing cells demonstrated that the proteolytically deknobbed Ad5-Zwt vector was functional and specific for receptor targeting.
Hong
Saw See
SS
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec and Institut Fédératif de Recherche IFR-62, 7, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France.
Magnusson
Maria K
MK
Henning
Petra
P
Lindholm
Leif
L
Boulanger
Pierre A
PA
D000256:Adenoviridae / Q000235:genetics*; D002460:Cell Line; D015951:Factor Xa / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D017510:Protein Folding; D011994:Recombinant Proteins; D011995:Recombination, Genetic; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics; D014161:Transduction, Genetic; D014771:Virion / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D014779:Virus Replication / Q000235:genetics
D008024:Ligands; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; D015951:Factor Xa