Abstract
<title>Abstract</title> <p>Background Adeno-associated viruses (AAVs) are non-glycosylated vectors that rely on cell-surface glycans such as heparan sulfate or galactose residues as co-receptors for cellular entry. Significant effort is currently directed toward improving AAV properties to overcome challenges that limit efficient transduction across diverse cell types and to reduce vector doses that can trigger toxicity. Most advances have focused on engineering genetic AAV variants, whereas the design of chimeric capsids through the attachment of functional molecules has been less explored. Because many enveloped viruses are heavily glycosylated, we sought to investigate whether controlled glycosylation of the AAV9 capsid could modulate tropism and enhance transduction efficiency. Results To do this, we generated a library of structurally diverse synthetic glycans, attached them to defined capsid residues, and examined how glycan structure, attachment site, and density influence vector internalization and transduction both in cell culture and in vivo. We modified two capsid residues—M471 and A591—located near the AAVR interaction surface, and found that the transduction efficiency of glycoengineered vectors is strongly dependent on both glycan structure and surface density. In general, high glycan density negatively affects AAV transduction, although the magnitude of this effect varies with the specific glycan. Capsid glycosylation also alters vector stability in vivo, with destabilization increasing alongside glycan density, except when sialylated glycans are used, which mitigate this effect. Notably, mannosylation at either M471 or A591 enhances AAV transduction in culture and in vivo, with modification at M471 showing the strongest benefit. Conclusion Collectively, these results demonstrate that AAV capsid glycoengineering can endow vectors with new functional properties. However, this strategy requires careful optimization, as glycan structure and density critically influence both infectivity and stability.</p>