Incorporation of tumor-targeting peptides into recombinant adeno-associated virus capsids.
The human parvovirus adeno-associated virus type 2 (AAV-2) possesses many features that make it an attractive vector for gene delivery in vivo. However, its broad host range may limit its usefulness and effectivity in several gene therapy applications in which transgene expression needs to be limited to a specific organ or cell type. In this study, we explored the possibility of directing recombinant AAV-2 transduction by incorporating targeting peptides previously isolated by in vivo phage display. Two putative loops within the AAV-2 capsid were examined as sites for incorporation of peptides. We tested the effects of deleting these loops and different strategies for the incorporation of several targeting peptides. The tumor-targeting sequence NGRAHA and a Myc epitope control were incorporated either as insertions or as replacements of the original capsid sequence. Viruses were assessed for packaging, accessibility of incorporated peptides, heparin binding, and transduction in a range of cell lines. Whereas recombinant viruses containing mutant capsid proteins were produced efficiently, transduction of several cell lines was significantly impaired for most modifications. However, certain mutants containing the peptide motif NGR, which binds CD13 (a receptor expressed in angiogenic vasculature and in many tumor cell lines), displayed an altered tropism toward cells expressing this receptor. Based on this work and previous studies, possible strategies for achieving in vivo targeting of recombinant AAV-2 are discussed.
Grifman
M
M
Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92186, USA.
Trepel
M
M
Speece
P
P
Gilbert
L B
LB
Arap
W
W
Pasqualini
R
R
Weitzman
M D
MD
D000595:Amino Acid Sequence; D015153:Blotting, Western; D018826:CD13 Antigens / Q000235:genetics* / Q000378:metabolism; D002213:Capsid / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D000229:Dependovirus / Q000235:genetics*; D016259:Genes, myc / Q000235:genetics; D049452:Green Fluorescent Proteins; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000235:genetics*; D017992:Parvovirus / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D008164:Luminescent Proteins; D009842:Oligopeptides; D011991:Receptors, Virus; D049452:Green Fluorescent Proteins; D006493:Heparin; D004247:DNA; D018826:CD13 Antigens