An Expanded Toolbox for Versatile Chemical Editing of Adeno‐Associated Virus

Abstract Site‐specific incorporation of noncanonical amino acids (ncAAs) into the adeno‐associated virus (AAV) capsid offers powerful opportunities to probe and engineer the properties of this leading vector for human gene therapy. However, this approach currently relies almost exclusively on a single azide‐containing ncAA, incorporated using the pyrrolysyl‐tRNA synthetase/tRNA pair. Here, we substantially broaden the scope of this technology by demonstrating successful incorporation of numerous ncAAs into AAV capsid using four different platforms, and by uncovering design principles that facilitate capsid tolerance to structurally diverse side chains. Using this expanded toolbox, we incorporate several different bioorthogonal conjugation handles into AAV for precise capsid modification. In particular, a tetrazine‐containing ncAA facilitated ultrafast conjugation of an anti‐HER2 nanobody to the capsid, creating conjugates that efficiently and selectively infect HER2+ cells. We further used this platform for optimized capsid PEGylation, which reduced its immunogenicity without compromising infectivity. Finally, we efficiently incorporated two distinct ncAAs into the AAV capsid, and subsequently labeled them orthogonally to attach two different entities. Together, these advances dramatically expand the chemistries that can be introduced into the AAV capsid, offering powerful new tools to both probe and engineer the properties of this promising gene therapy vector.