Adeno-associated virus (AAV) production

All steps were essentially followed as outlined in the manufacturer's protocol (AAV Purification Standard Kit; Cell BIolabs Inc.; https://www.cellbiolabs.com/sites/default/files/VPK-140-aav-purification-kit.pdf).

The following procedure was used for one 15 cm dish:

Production:

1. HEK 293T cells were passaged 2-3 times prior to transfection and then cultured until 70-80% confluency.
2. Co-transfect cells with the pAAV-DJ, pAAV-helper and the expression construct (Ca phosphate precipitation method).
3. After 60 hrs, replace medium with 2.5 mL TBS/1% Triton (TBS-T) and incubate for 3 min at room temperature. Gently shake the culture plate several times and harvest all media, including cells, in a sterile tube.
4. Centrifuge for 5 min at 3,000 rpm to pellet the transfected cells. Resuspend the cell pellet in 2.5 mL TBS-T.
5. Subject the cell suspension to three rounds of freeze/thaw cycles by alternating the tubes between a dry ice-ethanol bath and a 37°C water bath. Resuspend in 5 mL culture medium.
6. Collect the AAV supernatant by centrifugation at 10,000 x g for 10 minutes. Discard the pellet.
7. The viral supernatant was immediately purified.

Purification:

1. Add 1,000 U of benzonuclease (Millipore E1014) and incubate for 1 hr at 37°C. 
2. Filtrate mixture through a 0.2-mm low-protein binding filter (Millipore Milliguard). 
3. Load filtrate to AVB HiTrap sepharose columns (GE Healthcare) with a perfusion pump at a flow rate of 0.75 ml/min. 
4. Elute AAV particles with a low-pH elution buffer (0.2 M glycine-HCl, pH 2.7; 5 mL) 
5. Immediately neutralize with neutralization buffer (1 M Tris-HCl, pH 8.0; 5 mL). 
6. Buffer-exchange to PBS and concentrate with Amicon Ultra centrifugal filter units (molecular weight cut-off of 10 kDa; Millipore - retain 100 µL). 
7. Resuspend retained solution in 400 µL PBS
8. Reconcentrate if necessary (step 6)
9. Filter-sterilize by passing through a 0.2-mm low-protein binding filter. 
10. Viral titers were determined with a commercially available qPCR-based titration kit (e.g., from Takara Cat. 6233).