Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.
Mol Ther
; 16(8): 1467-73, 2008 Aug.
Article
en En
| MEDLINE
| ID: mdl-18560418
ABSTRACT
We have developed a highly specific and robust new method for labeling adeno-associated virus (AAV) vector particles with either biophysical probes or targeting ligands. Our approach uses the Escherichia coli enzyme biotin ligase (BirA), which ligates biotin to a 15-amino-acid biotin acceptor peptide (BAP) in a sequence-specific manner. In this study we demonstrate that by using a ketone isotere of biotin as a cofactor we can ligate this probe to BAP-modified AAV capsids. Because ketones are absent from AAV, BAP-modified AAV particles can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage modification methodology in the context of a mammalian cell lysate for the labeling of AAV vector particles with various fluorophores, and for the attachment of a synthetic cyclic arginine-glycine-aspartate (RGD) peptide (c(RGDfC)) to target integrin receptors that are present on neovasculature. Fluorophore labeling allowed the straightforward determination of intracellular particle distribution. Ligand conjugation mediated a significant increase in the transduction of endothelial cells in vitro, and permitted the intravascular targeting of AAV vectors to tumor-associated vasculature in vivo. These results suggest that this approach holds significant promise for future studies aimed at understanding and modifying AAV vector-cellular interactions.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Represoras
/
Dependovirus
/
Ligasas de Carbono-Nitrógeno
/
Proteínas de Escherichia coli
Límite:
Animals
/
Female
/
Humans
Idioma:
En
Revista:
Mol Ther
Asunto de la revista:
BIOLOGIA MOLECULAR
/
TERAPEUTICA
Año:
2008
Tipo del documento:
Article
País de afiliación:
Estados Unidos