1. ¹Gene Therapy Center, The Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Columbus, Ohio, USA
2. ²Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
3. ³Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
4. ⁴Division of Molecular Medicine, Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, Ohio, USA

Correspondence: Jeffrey S. Bartlett, Gene Therapy Center, WA3016, The Research Institute at Nationwide Children's Hospital, 700 Children's Drive, Columbus, Ohio 43205, USA. E-mail: Jeffrey.Bartlett@NationwideChildrens.org

Received 29 November 2007; Accepted 15 May 2008; Published online 17 June 2008.

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.