Synthesis and Concomitant Assembly of Adeno-Associated Virus- like Particles in Escherichia coli

Virus-like particles (VLPs) have been used for numerous pharmaceutical applications, particularly vaccination and drug delivery. Recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy, has been proposed as a vaccine scaffold, but high production costs limit its use. Here we establish intracellular production of AAV VLPs in Escherichia coli. VP3 capsid proteins of AAV serotype 5 (AAV5) were expressed, and VLPs were readily purified. The correct assembly was confirmed by ELISA with an intact-capsid AAV5 antibody and an AAVR domain as well as by atomic force microscopy. Biological functionality was demonstrated with a HeLa cell internalization assay. Coexpression of the assembly-activating protein (AAP) of AAV5 in E. coli improved capsid yield. This work provides the first evidence that AAV VLPs form in E. coli, opening new opportunities for production and exploration of AAV VLPs for biomedical applications.