The infectivity of AAV9 is influenced by the specific location and extent of chemically modified capsid residues

Several treatments for genetic diseases utilizing recombinant adeno-associated viruses (AAVs) have recently gained approval. However, the development of a greater number of therapeutic AAVs is constrained by certain limitations. While extensive efforts have concentrated on screening AAV genetic libraries, an alternative strategy involves modifying the AAV capsid by attaching various moieties. The capsid of AAV plays a pivotal role in transducing target cells and evading immune responses, making modifications a key avenue for engineering improved variants.

Careful control of both the degree and specific position of capsid modifications is crucial for optimizing transduction efficiency and minimizing undesired effects on transgene expression. These results underscore the importance of precision in AAV capsid modification to achieve optimal transduction efficiency while mitigating potential drawbacks on transgene expression.

In our study, we replaced specific AAV9 capsid residues with an unnatural amino acid bearing a bioorthogonal group, identifying four positions with no adverse impact on production. Utilizing click chemistry, we attached varying proportions of Cy5.5 to these positions, allowing us to assess the impact of these modifications on AAV9 infectivity in cultured cells. Our findings reveal that both the position and degree of capsid modification significantly affect AAV transduction. While higher amounts of attached molecules lead to an increased number of AAV genomes within cells, this does not positively impact transgene expression. Conversely, a negative impact on transgene expression is observed when the AAV capsid is highly modified, with the degree of this effect associated with the modified residue.