Enhanced cellular secretion of AAV2 by expression of foreign viral envelope proteins

•Viral envelope proteins can enhance cellular secretion of non-enveloped virus.•Enhanced secretion of rAAV2 by VSVG was via exosome-mediated pathway.•Secreted rAAV2 particles were susceptible to AAV neutralizing antibodies. Recombinant adeno-associated virus (rAAV), a non-enveloped virus, is widely used in gene therapy clinical trials because it does not cause human disease, transduces both dividing and non-dividing cells, and mediates stable transgene expression for years in post-mitotic tissue. Extension of clinical use of rAAV is, however, considerably hampered by difficulties involved in large-scale production of the virus particles. For several serotypes of rAAV these difficulties often arise from the fact that assembled virus particles mainly stay inside of packaging cells, inevitably requiring lysis of cells to harvest virus particles and consequentially complicating downstream purification processes. Here, we show that introduction of foreign viral envelope protein genes, encoding for either VSVG or rabiesG, into packaging cells can remarkably enhance cellular secretion of rAAV2, the AAV serotype most often used in clinical applications. In the presence of the foreign genes, up to 49% of transducing rAAV2 particles were secreted. However, such great enhancement was not observed for rAAV3. Our experimental tests with exosome inhibitors indicated that VSVG-mediated cellular secretion of rAAV2, unlike rabiesG-mediated one, heavily relies upon cellular pathways involving exosomes. Ultimately, an improved understanding of rAAV secretion mechanisms may simplify the production and purification processes for large-scale batches of the virus.