Adeno-associated viruses (AAVs) have been employed for treating several monogenic diseases, and some AAV-based therapies like Luxturna and Zolgensma have been approved by the FDA. Nonetheless, the use of AAVs has limitations, such as pre-existing neutralizing antibodies, high-dose toxicity, low virus transduction specificity, and potential immune response induction. Therefore, it is crucial to develop novel AAV versions to overcome these limitations. Our team is focused on generating chimeric viral particles linking molecules with desirable characteristics to AAV9 capsid to create new variants with improved properties.

We incorporated the unnatural amino acid azidolysine at specific positions of the AAV9 capsid by replacing residues in poorly conserved sequences between AAV serotypes. We successfully produced AAV9 mutants with azidolysine at five different positions on the capsid, achieving similar titers to the AAV9 WT. The presence of azidolysine on the capsid surface allows us to attach interesting molecules through simple "Click Chemistry" reactions. We evaluated the sensitivity of the most promising mutants to modification using the fluorescent molecule CY5.5 with a DBCO group and their infectivity after modification. These findings will help us identify the best candidates to use with other molecules that can provide AAV9 with new characteristics, such as glycans, aptamers, or superparamagnetic nanoparticles.