Assessment of Enzyme-Mediated Modulation of rAAV Vectors: Effects on Cellular Transduction and Capsid Stability

Recombinant adeno-associated virus (rAAV) vectors are widely used in gene therapy because of their low immunogenicity. However, pre-existing immunity remains a major challenge, since neutralizing antibodies can effectively lower transduction. The rAAV capsid surface serotype is a critical quality attribute for gene therapy vectors. The capsid defines vector stability and cellular transduction through the capsid’s surface protein interactions with cellular receptors. While post-translational modifications such as deamidation, phosphorylation, and acetylation impact potency and stability, there remains a need to better understand glycosylation. Although low in abundance, even limited N-linked glycosylation may influence transduction efficiency and immune evasion. In this study, N-linked glycan profiles was investigated focusing on endo- and exo-glycosidases for modulating rAAV transduction and stability. Our results indicate that high mannose glycans may shield capsid sites from deamidation, preserving 3–6% transduction efficacy. Like monoclonal antibodies, we expected N-linked glycans to influence rAAV aggregation and stability, but saw limited impacts potentially due to glycan abundance, heterogeneity, or robust formulation conditions. These findings highlight glycosylation’s nuanced role in rAAV biology and demonstrate glycan engineering as a potential strategy to enhance gene therapy vector performance.