727. Characterizing the Adeno-Associated Virus 1 Sialic Receptor Binding Site and Its Overlap with Antigenic Epitopes

Adeno-associated viruses (AAVs) are being development as gene delivery vectors and have shown promise in several clinical trials. And an AAV1-based vector has been approved by the European Commission for the treatment of lipoprotein lipase deficiency disease. However, limitations in tissue transduction specificity as well as neutralization by pre-existing antibodies remain are two major obstacles. To overcome these problems, it is important to characterize functional capsid regions that dictate virus binding to cellular receptors during infection and the potential overlap(s) of these regions with capsid antigenic epitopes. In this study, the sialic acid (SIA) binding site on AAV1 was determined by solving the structure of the AAV1-SIA complex using X-ray crystallography. Residues that form the SIA binding pocket are identical between AAV1 and AAV6; hence, structurally predicted SIA binding sites were mutated on both serotypes to confirm their role in the SIA interaction. Binding and transduction assays with these mutants in CHO cell lines Pro5, Lec2, Lec8, and Lec1, which display different terminal glycans, confirmed the structurally mapped binding pocket. Furthermore, native dot blot showed that several of the AAV1 SIA binding mutants can escape from antibody recognition. This finding is consistent with the overlap between the SIA binding site and the previously mapped AAV1 epitopes. Significantly, a glycan binding mutant with slightly reduced transduction ability is able to escape from antibody recognition. This study identifies an overlap between receptor recognition and antibody reactivity and provides amino acid level information for rational capsid engineering of AAV vectors for improved therapeutic efficacy.