Site-Specific Chemical Conjugation of Recombinant Proteins onto the AAV Capsid.

Adeno-associated virus (AAV) is the leading vector for developing in vivo gene therapies. However, native AAV capsids exhibit limited control over tissue tropism, often necessitating high doses that lead to off-target effects and reduced therapeutic efficacy. Here, we present a robust and generalizable strategy to posttranslationally conjugate recombinant proteins, including full-length antibodies, onto the AAV capsid with precise control over the site and stoichiometry of attachment. By genetically encoding a bioorthogonal chemical handle into select capsid proteins using noncanonical amino acid (ncAA) mutagenesis, we enable selective labeling of the virus capsid with recombinant proteins using bifunctional linkers. This modular approach allows for subsequent rapid conjugation of site-specifically modified recombinant proteins through ultrafast click chemistry. Our method preserves AAV integrity and infectivity, and enables programmable retargeting of AAV tropism toward disease-relevant receptors. This platform provides a powerful approach for engineering next-generation AAV vectors with customizable targeting profiles for diverse therapeutic applications.