Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid.
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.
Vellinga
J
J
Department of Molecular Cell Biology, Leiden University Medical Center, Einthovenweg, Leiden, The Netherlands.
de Vrij
J
J
Myhre
S
S
Uil
T
T
Martineau
P
P
Lindholm
L
L
Hoeben
R C
RC
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
D036022:Capsid Proteins; D007128:Immunoglobulin Fragments; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D001616:beta-Galactosidase