Method for producing recombinant adeno-associated virus

A method of producing recombinant adeno-associated virus (rAAV), comprising the following steps:

• i) cotransducing host cells with a transduction solution comprising the following recombinant baculoviruses:
• Bac-lacZ, Bac-RC and Bac-Helper;
• and
• ii) culturing the cotransduced host cells resulting from step i) in a medium,
• wherein said host cells are mammalian cells;
• said Bac-lacZ is a recombinant baculovirus harboring a reporter gene flanked by adeno-associated virus serotype 2 (AAV-2) inverted terminal repeats (ITRs);
• said Bac-RC is a recombinant baculovirus harboring AAV-2 rep and cap genes;
• and said Bac-Helper is a recombinant baculovirus harboring adenovirus E2A, E4, and VA RNA genes,
• wherein the host cells in step i) are immobilized on carriers and the cotransducing in step i) comprises submerging the host cells on the carriers in the transduction solution and exposing the host cells on the carriers to air alternately,
• wherein the culturing in step ii) comprises submerging the cotransduced host cells on the carriers resulting from step i) in the medium and exposing the cotransduced host cells on carriers to air alternately,
• wherein the medium is contained in a first chamber, the carriers are contained in a second chamber connected to the first chamber, and the first chamber is compressed and relaxed so as to submerge the cotransduced host cells on the carriers in the medium and expose the host cells on the carriers to air alternately, and
• wherein the culturing in step ii) further comprises a perfusion operation which is performed throughout the culturing period, said perfusion operation comprising intermittently feeding a fresh medium, which is the same as the medium for culturing the cotransduced host cells, to the cotransduced host cells on the carriers, and intermittently withdrawing the medium from the first chamber in an amount equivalent to the feeding amount of the fresh medium, so that a fixed amount of the medium submerging the cotransduced host cells on the carriers is maintained.