GFP Adeno-Associated Virus ( AAV-I-587 )

Insertion of Peptides into the Common VP3 Region When Girod et al. reported the first successful capsid modification for re-targeting in 1999, no crystal structure for AAV capsids was available, and possible insertion sites were predicted based upon the crystal structure of canine parvovirus (CPV). Of the six candidate insertion sites (I) of AAV2 (I-261, I-381, I-447, I-534, I-573, and I-587), three were suitable for surface display of the β1 integrin-binding model ligand L14 (QAGTFALRGDNPQG). Ultimately, however, only insertion of the L14 peptide into I-587, between amino acid residues asparagine (N) 587 and arginine (R) 588, allowed target receptor-mediated cell transduction. Target cells were transduced even in the presence of heparin, a soluble analog of heparan sulfate proteoglycans (HSPGs), the primary receptor for AAV2. Insertion of the peptide at I-587 alters the spacing between R585 and R588, disrupting the AAV2 HSPG binding motif. Mapping I-587 onto the crystal structure of AAV2 confirmed that the insertion site is indeed located at the apex of the loop. Specifically, I-587 is part of VR-VIII, which forms the top of the second tallest of three protrusions on the 3-fold symmetry axis. A series of subsequent studies confirmed the potential of this insertion site for capsid engineering, including studies defining peptide features for vector retargeting. Thus, the AAV2 I-587 insertion site allows genetic modifications of the capsid without interfering with capsid assembly or genome packaging, accepts peptides of up to a size of 34 amino acids, displays the foreign sequence in such a way that peptides can interact with target cell surface molecules, and enables vector re-targeting in a single step because the AAV2 primary receptor binding motif is modified upon insertion of the novel targeting ligand.