Using Unnatural Amino Acid Incorporation to Modify and Manipulate Adeno-Associated Virus

Abstract

Adeno-Associated Virus (AAV) has been developed into a powerful therapeutic tool - in the last ten years it has acted as a gene-delivery vehicle in several approved therapeutics and many more therapeutics on trial. Despite extensive research, gaps in our understanding of AAV’s infectious cycle still exist, and further development is needed for the creation of improved gene therapy vectors. Technology to incorporate Unnatural Amino Acids (UAAs) into the AAV capsid has recently been developed, and could aid in both furthering our understanding of AAV’s biology and in the therapeutic advancement of AAV. In this work, we demonstrate how the functionalization of the AAV capsid using UAA incorporation can advance our control over the AAV capsid and aid in probing and manipulating AAV biology. We describe our use UAA incorporation to place a bio-orthogonal reactive handle into AAV’s capsid followed by functionalization with a targeting moiety and demonstrate the unprecedented amount of control that UAA incorporation provides in the creation of a functional virus conjugate. We are able to control both the precise placement and the stoichiometry of the targeting moiety on the AAV capsid, providing a platform that, for the first time, can undergo rigorous optimization analogous to that which medicinal chemists put small molecules through. We also describe the creation of a new platform to site-specifically modify the AAV capsid using cysteine incorporation, a technique that retains the ability to site-specifically modify the capsid as UAA incorporation does, but does not require the excess machinery that UAA incorporation requires. Next we discuss the incorporation of a photocaging amino acid, NBK, into the AAV capsid. Using NBK, we were able to effectively block AAV’s primary binding interaction with Heparan Sulfate Proteoglycan (HSPG) and control the timing of AAV infection using light to chemically remove the photo-protecting group. While photocaging the HSPG interaction is only a proof of concept, it demonstrates the remarkable amount of control that UAA incorporation affords, and lends insight to what could be accomplished using the functionalities that can be placed on the AAV capsid with UAAs.