title: Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid.
abstract: Recombinant adenoviruses are frequently used as gene transfer vehicles for therapeutic gene delivery. Strategies to amend their tropism include the incorporation of polypeptides with high affinity for cellular receptors. Single-chain antibodies have a great potential to achieve such cell type specificity. In this study, we evaluated the efficiency of incorporation of a single-chain antibody fused with the adenovirus minor capsid protein IX in the capsid of adenovirus type 5 vectors. To this end, the codons for the single-chain antibody fragments (scFv) 13R4 were fused with those encoding of pIX via a 75-Angstrom spacer sequence. The 13R4 is a hyper-stable single-chain antibody directed against beta-galactosidase, which was selected for its capacity to fold correctly in a reducing environment such as the cytoplasm. A lentiviral vector was used to stably express the pIX.flag.75.13R4.MYC.HIS fusion gene in 911 helper cells. Upon propagation of pIX-gene deleted human adenovirus-5 vectors on these cells, the pIX-fusion protein was efficiently incorporated in the capsid. Here, the 13R4 scFv was functional as was evident from its capacity to bind its ligand beta-galactosidase. These data demonstrate that the minor capsid protein IX can be used as an anchor for incorporation of single-chain antibodies in the capsids of adenovirus vectors.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg, Leiden, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Vellinga
authors.forename: J
authors.initials: J
authors.lastname: de Vrij
authors.forename: S
authors.initials: S
authors.lastname: Myhre
authors.forename: T
authors.initials: T
authors.lastname: Uil
authors.forename: P
authors.initials: P
authors.lastname: Martineau
authors.forename: L
authors.initials: L
authors.lastname: Lindholm
authors.forename: R C
authors.initials: RC
authors.lastname: Hoeben
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000648:ultrastructure; D015153:Blotting, Western / Q000379:methods; D036022:Capsid Proteins / Q000235:genetics*; D002461:Cell Line, Transformed; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics; D016253:Microscopy, Immunoelectron; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D001616:beta-Galactosidase