Adeno-associated virus (AAV) vectors have attracted considerable interest because of its great premise as a vector for human gene therapy. However, their applicability is limited due to the restricted range of cells that they can efficiently transduce. Although alternative AAV serotypes might be available for enhancing gene delivery to certain cells/tissues, this does not provide selective tropism of virus for specific target cells or tissues. Therefore, it is desirable to redirect AAV vector tropism that specifically transduces selected target cells in vivo. Currently, the majority of targeting efforts rely upon genetic modification of AAV capsid proteins and chemical conjugation utilizing the reactivity of functional groups in order to introduce targeting ligands onto AAV. However, genetic insertion of targeting peptide motifs into AAV capsid usually results in low production yield, dramatic reduction of vector titer or significant drop of DNA packing ability. Moreover, the lysine modification, which is wildly used for chemical conjugation of targeting ligands onto the viral surface, has been known to be often problematic because lysine residues are likely involved in conformational change of virus and/or host-virus interactions that are necessary for infection.
To overcome these limitations, we have developed a new method for site-specific modification of AAV2 in order to generate a targeted AAV2 vector. This approach involves the insertion of a genetically encoded aldehyde tag (13-residues: LCTPSRAALLTGR) onto AAV2 capsids, which can be metabolically modified to generate an aldehyde group as a unique chemical handle for further site-specific introduction of targeting ligands without causing a significant loss of viral titer. We demonstrated that this aldehyde tag enables covalent attachment of hydrazide-functionalized molecules including antibodies and peptides to AAV2 vector in a site-specific manner. It showed that antibody conjugation to AAV2 could significantly enhance viral transduction in both permissive and non-permissive cell lines expressing alternative cell-surface receptors. In addition, RGD-peptide conjugated AAV2 exhibited tumor targeting in vitro. These results demonstrated a general and efficient means for targeting AAV vectors.