Analysis of Protein Carrier AAV by LC-MS/MS

AAV is widely used for efficient delivery of DNA payloads. The extent to which the AAV capsid can be used to deliver a protein payload is unexplored. Here, we report engineered AAV capsids that directly package proteins – Protein Carrier AAV (pcAAV). Nanobodies inserted into the interior of the capsid mediate packaging of a cognate protein, including Green Fluorescent Protein (GFP), Streptococcus pyogenes Cas9, Cre recombinase, and the engineered peroxidase APEX2. We show that protein packaging efficiency is affected by the nanobody insertion position, the capsid protein isoform into which the nanobody is inserted, and the subcellular localization of the packaged protein during recombinant AAV capsid production; each of these factors can be rationally engineered to optimize protein packaging efficiency. We demonstrate that protein packaged within pcAAV retain their enzymatic activity and that pcAAV can bind and enter the cell to deliver the protein payload. Establishing pcAAV as a protein delivery platform expands the utility of AAV as a therapeutic and research tool.