Abstract
Current adeno-associated virus (AAV) gene therapies are limited by a lack of tissue-specificity, leading to toxic off-target impacts and increased therapeutic costs. More target-specific AAV vectors can offer safer and less expensive therapies. Antibody-like proteins can be used to retarget AAV vectors to unique cell-surface receptors. However, fusing such proteins onto the delicate AAV capsid proteins is complicated; only small and well-folded proteins are tolerated and the architecture of such fusions are highly restricted. Here, we report a versatile chemical strategy to covalently attach recombinant proteins onto the AAV capsid, using a combination of genetic code expansion (GCE) and bioorthogonal conjugation chemistry. This method is efficient, allows excellent control over the site of attachment, and the number of proteins attached on the AAV capsid. Systematic optimization of these parameters allowed us to generate highly active AAV2-anti-HER2 nanobody conjugates that significantly outperform their counterparts generated through genetic fusion. This conjugate exhibited excellent efficiency and selectivity toward HER2+ cancer cells both in vitro and in vivo. Furthermore, we demonstrate that our strategy can be used to attach diverse recombinant proteins, including superfolder green fluorescent protein (sfGFP) and a full-length antibody, creating novel opportunities to engineer AAV.