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.