Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations.
BACKGROUND
Genetic modification of capsid proteins by peptide insertion has created the possibility of using adeno-associated viral (AAV) vectors for receptor specific gene transfer (AAV targeting). The most common site used for insertion in AAV serotype 2 capsids are amino acid positions 587 and 588 located at the second highest capsid protrusion. Reasoning that peptide insertions at the most exposed position augments target receptor interaction, we explored position 453 as a new insertion site.


METHODS
Position 453 was identified in silico. Capsid mutants carrying the model ligand RGD-4C in position 453 with and without R585A/R588A substitutions were compared with respective mutants carrying the ligand in position 587. The accessibility of the inserted ligand was determined by an enzyme-linked immunosorbent assay, whereas the transduction efficiency and specificity of receptor binding were assayed by gene transfer and competition experiments, respectively. Vector biodistribution was determined in mice by quantitative polymerase chain reaction analysis.


RESULTS
Initially, RGD-4C, inserted at position 453, failed to efficiently bind its target receptor. R585 and R588, located at the neighboring peak and known to mediate primary receptor binding, were identified as interfering residues. R585A and R588A substitutions rendered position 453 mutants superior to those with the ligand in position 587 in target receptor binding and cell transduction efficiency. The in vivo biodistribution was independent of the insertion site, but directed by the inserted ligand when primary receptor binding was avoided.


CONCLUSIONS
Position 453 emerged as a prominent site for the development of targeting mutants. Furthermore, we show for the first time that linearly distant residues can be critical for the efficiency of inserted peptide ligands.
Boucas
Jorge
J
Department I of Internal Medicine, University of Cologne, Cologne, Germany.
Lux
Kerstin
K
Huber
Anke
A
Schievenbusch
Stephanie
S
von Freyend
Miriam John
MJ
Perabo
Luca
L
Quadt-Humme
Sibille
S
Odenthal
Margarete
M
Hallek
Michael
M
Büning
Hildegard
H
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics* / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics*; D014161:Transduction, Genetic
D036022:Capsid Proteins; D009842:Oligopeptides