Production and Chemoselective Modification of Adeno-Associated Virus Site-Specifically Incorporating an Unnatural Amino Acid Residue into Its Capsid.

The ability to modify the capsid proteins of human viruses is desirable both for installing probes to study their structure and function, and to attach retargeting agents to engineer viral infectivity. However, the installation of such capsid modifications currently faces two major challenges: (1) The complex and delicate capsid proteins often do not tolerate large modifications, and (2) capsid proteins are composed of the 20 canonical amino acids, precluding site-specific chemical modification of the virus. Here, we describe a technology for generating adeno-associated virus (AAV) while incorporating an unnatural amino acid (UAA) into specific sites of the virus capsid. Incorporation of this UAA is generally tolerated well, presumably due to its small structural footprint. The resulting virus can be precisely functionalized at the site of UAA incorporation using chemoselective conjugation strategies targeted toward the azido side chain of this UAA. This technology provides a powerful way to modify AAV with unprecedented precision to both probe and engineer its entry process.