Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

All currently identified primary receptors of adeno-associated virus (AAV) are glycans. Depending on the AAV serotype, these carbohydrates range from heparan sulfate proteoglycans (HSPG), through glycans with terminal alpha 2-3 or alpha 2-6 sialic acids, to terminal galactose moieties. Receptor identification has largely relied on binding to natural compounds, defined glycan-presenting cell lines, or enzyme-mediated glycan modifications. Here, we describe a comparative binding analysis of highly purified, fluorescent-dye-labeled AAV vectors of various serotypes on arrays displaying over 600 different glycans and on a specialized array with natural and synthetic heparins. Few glycans bind AAV specifically in a serotype-dependent manner. Differential glycan binding was detected for the described sialic acid-binding AAV serotypes 1, 6, 5, and 4. The natural heparin binding serotypes AAV2, -3, -6, and -13 displayed differential binding to selected synthetic heparins. AAV7, -8, -rh.10, and -12 did not bind to any of the glycans present on the arrays. For discrimination of AAV serotypes 1 to 6 and 13, minimal binding moieties are identified. This is the first study to differentiate the natural mixed heparin binding AAV serotypes 2, 3, 6, and 13 by differential binding to specific synthetic heparins. Also, sialic acid binding AAVs display differential glycan binding specificities. The findings are relevant for further dissection of AAV host cell interaction. Moreover, the definition of single AAV-discriminating glycan binders opens the possibility for glycan microarray-based discrimination of AAV serotypes in gene therapy.