Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase.
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.
Gene Therapy Center, The Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Columbus, Ohio, USA.
Matthew D
MD
Stachler
Irwin
I
Chen
Alice Y
AY
Ting
Jeffrey S
JS
Bartlett
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D000229:Dependovirus / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D015336:Molecular Probe Techniques; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010456:Peptides, Cyclic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D008164:Luminescent Proteins; D010456:Peptides, Cyclic; D012097:Repressor Proteins; C507169:cyclic arginine-glycine-aspartic acid peptide; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli