Mechanisms of capsid protein nuclear import across divergent adeno-associated virus serotypes

Adeno-associated viruses (AAVs) are widely recognized as effective gene therapy vectors due to their non-pathogenic nature, tissue-specific targeting, and customizable engineering potential. With over 150 serotype variants across 12 recognized types, AAVs have become pivotal tools in addressing genetic disorders. Central to their effectiveness is the understanding of AAV capsid interactions facilitating nuclear localization, crucial for therapeutic transgene expression. Despite extensive research, the precise mechanisms governing AAV nuclear import remained elusive. Here, we present a comprehensive investigation elucidating these mechanisms and their implications for vector design. We hypothesized AAVs utilize the classical importin α (IMPα)-dependent pathway for nuclear entry, with specificity between AAV serotypes and IMPα isoforms. Biochemical assays confirmed interactions between AAV variants and IMPα isoforms, implicating the classical pathway. Cellular studies further supported this, revealing IMPα dependency for AAV VP1 nuclear localization. Structural analyses unveiled bipartite nuclear localization signals (NLSs) within AAV capsids, with AAV3 exhibiting a monopartite NLS. Notably, AAV serotypes exhibited differential binding affinities for IMPα isoforms, suggesting potential for vector optimization. Substitution of NLS regions between AAV serotypes could provide new capsid engineering target and possibly enhance transgene expression or reduce required vector doses, mitigating toxicity and immune responses. Future investigations should explore the impact of NLS modifications on vector efficiency and toxicity. Additionally, comprehensive profiling of AAV serotypes and non-human variants is warranted for a thorough understanding of vector-host interactions. Notably, non-human AAV variants present novel opportunities for vector design, owing to their distinct NLSs and IMPα affinities. In conclusion, our study provides unprecedented insights into AAV capsid interactions, offering valuable targets for optimizing gene therapy vectors and overcoming current limitations in clinical applications.