A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

The details of the reagents, plasmids, and equipment used in the study are listed in the Table of Materials. The composition of the buffers used is provided in Supplementary File 1.

1. Plasmid preparation

1. Transform plasmids (pAAV-GOI, pHelper, pAAV-Cap) in E. coli.
   NOTE: Any E.coli strain can be used for plasmid amplification. For some plasmids, special competent cells such as NEB stable can improve the plasmid yield. The three plasmids used in this protocol are pAAV-GOI: pAAV-CMV-GFP, pHelper: pAdDeltaF6, and pAAV-Cap: pAAV-RC6.
2. Grow bacteria in LB media containing the appropriate selective antibiotics in optimal volume at 37 °C for 16-18 h.
   NOTE: When using NEB stable cells, culture at 30 °C for 18-20 h.
3. Harvest plasmid DNA by centrifuging the E. coli. culture medium at 4000 x g, 4 °C, for 15 min.
4. Carefully pour the supernatant liquid from the centrifuge bottle into a waste container and purify the plasmid DNA from the pellet with a large-scale endotoxin-free plasmid purification kit.
   NOTE: Ensure to pour it slowly and avoid splashing.
5. Measure the DNA concentration and purity using a spectrophotometer.
   NOTE: Check the DNA purity by checking OD₂₆₀/OD₂₈₀ ratio. The ratio for pure DNA is about 1.8. DNA concentration should be above 1 µg/µL for the later co-transfection step. Make bacterial glycerol stocks by adding 500 µL of the overnight culture to 500 µL of 50% glycerol in a cryovial and store at -80 °C.

2. Preparing HEK293T cells

1. Seed HEK293T cells with DMEM high glucose media containing 10% FBS into a 10-layer cell factory (CF10) and let the cells grow in the incubator overnight.
   NOTE: Passages of the 293T cells should be less than 10 to have the optimal condition for robust AAV production. If possible, do not add antibiotics to the culture media at this time. CF10 has approximately 42 times the growth surface area of a 150 mm cell culture dish. Seed the same cell density in a 150 mm cell culture dish to allow checking the cell growth under a microscope. Prepare 1075 mL cell suspension, add 25 mL cell suspension to a 150 mm dish, and add the remaining into the CF10.
2. Check cell confluency the next day before the transfection.
   NOTE: Cells should reach 80%-90% confluency at the time of transfection by observing under a microscope.

3. Triple-transfection of AAV plasmids

1. Calculate the amount of each plasmid needed (pAAV-GOI, pHelper, pAAV-serotype) to have a 1.2: 1: 1 molar ratio with 2.5-5 mg of total DNA per CF10.
   NOTE: The three plasmids' molar ratio might be variable for the optimal transfection efficacy. It is better to test it in a small-scale setting before moving to this CF10 setting. pAAV-CMV-GFP, pAdDeltaF6, and pAAV-RC6 are used to make the AAV6-CMV-GFP virus as an example. The PEI calculator formula is listed in Table 1.
2. Aliquot 350 mL of OptiMEM into a sterile 500 mL bottle.
3. Add all three plasmid DNA into the bottle containing the Opti-MEM. Mix well.
4. Add 15 mL of 1 mg/mL polyethylenimine (PEI) solution (1:3 µg of DNA to µg PEI ratio). Shake the OptiMEM/DNA/PEI mixture up and down vigorously for 30 s.
   NOTE: It is okay to make bubbles.
5. Incubate the mixture at room temperature for 10-15 min.
   NOTE: Longer incubation can reduce transfection efficiency.
6. Add the OptiMEM/DNA/PEI solution to the 1 L bottle containing 700 mL of DMEM low glucose with 10 mM HEPES and 2% FBS. Mix well.
   NOTE: In this study, the low glucose in the DMEM showed better AAV vector production after co-transfection. The addition of HEPES helps to maintain the cells in a better condition. Mix by rotating the bottle slowly. Avoid creating too many bubbles.
7. Take the CF10 out of the incubator and vacuum the media out into a waste container.
   NOTE: Put down the CF10 on the surface inside the hood and gradually lift one side up, slowly vacuum the media out without detaching the cells.
8. Carefully add the transfectant mixture to the CF10. Ensure that all ten layers are covered with media.
   NOTE: Add 25 mL of transfectant mixture into the 150 mm dish. Monitor the cells daily until the harvest. Add the remaining transfectant mixture into the CF10 by pouring slowly. Rotate the CF10 very carefully and ensure equal volume for each layer.
9. Return the CF10 to the incubator for 72-96 h.
   NOTE: Check the 150 mm monitor dish to decide when to harvest the AAV vectors. When the cells become very easily detached with a full load of AAV, it is time to harvest.

4. Harvesting AAV vectors

1. Harvest the virus 3-4 days after transfection by shaking the CF10 vigorously. Pour the media into the 250 mL conical tubes and spin down the virus at 4000 x g for 20 min at 4 °C.
   NOTE: For the AAV6 serotype used in this protocol, 80% AAV will remain mostly bound to cellular material in the pellet. And the 20% AAV were secreted into the media. These proportions may differ for other AAV serotypes.
2. Filter clarified supernatant with 0.45 µm filter unit and save it for precipitation.
3. Rinse the CF10 with 500 mL of DPBS 1x buffer and centrifuge at 4000 x g for 20 min at 4 °C.
4. Discard the supernatant using a vacuum system and an aspirating pipette. Add 20 mL of AAV lysis buffer per CF10 at -80 °C until purification.
   NOTE: Transfer AAV lysate to a 50 mL conical tube for later AAV extraction.
5. Take out the filtered supernatant, add 40% PEG-2.5 M NaCl solution for a final concentration of 8% PEG, and incubate in a cold room on an orbital rotator for at least 3 h or overnight.
   NOTE: For 100 mL supernatant, add 25 mL 40% PEG-2.5 M NaCl solution.
6. Precipitate the virus by centrifuging at 4000 x g for 30 min at 4 °C.
   NOTE: Transfer the mixture to 250 mL conical tubes for centrifugation.
7. Aspirate to remove supernatant and add 5-10 mL of AAV HEPES resuspension buffer to suspend the pellets. Transfer into a 50 mL conical tube to continue the downstream purification, or store it at -80 °C.

5. AAV extraction

1. Thaw the virus pellet in a 37 °C water bath for 10-15 min with a shaker.
   NOTE: Vortex the AAV lysate to fully melt.
2. Freeze in -80 °C 100% ethanol bath for 20-30 min.
   NOTE: Alternatively, the freeze cycle can be done with a cold 100% ethanol beaker surrounded by dry ice.
3. Perform freeze/thaw 3-4 times, and vortex in between if necessary.
   NOTE: This cycle can be paused at the freeze step. Store the AAV lysate at -80 °C until the downstream work.
4. Thaw AAV lysate (from step 5.3) and resuspended AAV (from step 4.7)” in a 37 °C water bath for 10-15 min with a shaker, and vortex.
   NOTE: Ensure to melt completely and mix it.
5. Add Benzonase nuclease (250 units/µL) to AAV lysate and resuspended AAV for a final concentration of 50 units/mL. Vortex the solution.
6. Incubate in a 37 °C water bath with shaking for 30 min.
   NOTE: Take out 10% sodium deoxycholate aliquots in a water bath to allow time to dissolve, warm up, and mix thoroughly.
7. Add 10% sodium deoxycholate for a final concentration of 0.5% and incubate at 37 °C with a shaker on for 30 min.
   NOTE: Sodium deoxycholate was used to enhance AAV release from the cells.
8. Distribute the raw AAV solution in 2 mL centrifuge tubes and centrifuge at 14,000 x g for 10 min at 4 °C.
9. Transfer the supernatant into a 50 mL conical tube.
   NOTE: Use a 1 mL pipette to transfer the supernatant. Discard the pellets.
10. Add an equal amount of chloroform and extract AAV by vortexing for 1-2 min.
    NOTE: The solution should become opaque, white, milk-like.
11. Transfer the milky solution into 2 mL centrifuge tubes and distribute evenly. Centrifuge at 14,000 x g for 10 min at 4 °C.
12. Carefully pipette out the top pink layer and transfer it in a new 50 mL conical tube.
    NOTE: Do not disturb the chloroform/protein layers.
13. Measure the final volume using a pipette. According to the raw AAV-PEG-Sulfate volume chart (Table 2), mix appropriate volumes of 50% (NH₄)₂SO₄ and 40% PEG solution. Vortex for 2 min.
14. Transfer the mixture to 2 mL centrifuge tubes to distribute evenly. Centrifuge at 14,000 x g for 10 min at 4 °C
15. Collect the bottom layer by using a 22 G needle and a 3 mL syringe. Collect the AAV in a 50 mL conical tube and store it at 4 °C for later iodixanol gradients ultracentrifugation.

6. AAV purification by iodixanol gradient ultracentrifugation

1. Prepare the iodixanol gradients freshly before loading the AAV according to the number of ultracentrifugation tubes used (Table 3).
   NOTE: Phenol red was used to observe the layers.
2. Overlay each solution into a round-top polypropylene ultracentrifuge quick-seal tube slowly using a 10 mL syringe attached with a 16 G punted long needle. Avoid bubbles.
3. Carefully add up to 17 mL of AAV solution on top of the gradient with a 22 G syringe. Use AAV dialysis buffer to top off the tube.
   NOTE: Add the AAV solution by loading drops against the wall in the ultracentrifuge tube. Don't disturb the gradient layers.
4. Seal the ultracentrifuge tubes with an electric sealer.
   NOTE: Balance the tubes before sending them to ultracentrifuge with ±0.2 g difference.
5. Centrifuge at 350,000 x g for 2 h in a Ti70 rotor at 4 °C.
6. Carefully remove the tubes from the rotor and place them in the ultracentrifuge tubes rack.
   NOTE: Ensure that the gradients are not disturbed.
7. Collect AAV fractions following the steps below:
   1. Stabilize the tube on a stand holder.
   2. Puncture the ultracentrifuge tubes slightly below the 40%-60% interface with a 19 G needle attached to a 10 mL syringe.
      NOTE: The opening of the needle should be up, facing the 40% gradient.
   3. Punch a hole in the top of the tube with a 16 G needle.
   4. Collect up to 5 mL per tube. Avoid collecting the protein contamination at the 25%-40% interface.
   5. Repeat for each ultracentrifuge tube.
      NOTE: Transfer the AAV fractions to a 50 mL conical tube.

7. Second round iodixanol gradients ultracentrifugation

NOTE: This step is optional. This step is to reduce the empty AAV ratio for a higher quality full AAV capsid.

1. Dilute AAV >1:1 with AAV dialysis buffer.
   NOTE: AAV collected from the first round of ultracentrifugation was at 40% iodixanol layer. It should be diluted by at least 50% to be able to be loaded on top of the 30% iodixanol layer.
2. Overlay each solution (Table 4) into an ultracentrifugation tube using a 16 G punted needle and slowly attach a 10 mL syringe. Avoid bubbles.
3. Carefully add 20 mL of diluted AAV solution on top of the gradient with a 22 G syringe. Use AAV dialysis buffer to top off the tube.
4. Repeat the steps 6.4 to 6.7.

8. AAV dialysis and concentration

1. Transfer the AAV virus into the dialysis cassette using a new 18 G needle and 10 mL syringe. Slowly inject the virus into the cassette and remove air from it.
2. Add a stir bar to the beaker containing AAV dialysis buffer and place it on a stir plate.
3. Put the dialysis cassette into the dialysis buffer with a float buoy. Stir at 4 °C. Change 2-3 times the AAV dialysis buffer.
   NOTE: Use enough buffer to allow the cassette to float and perform the buffer exchange. Cover the beaker with aluminum foil.
4. Using a 10 mL syringe attached with an 18 G needle, collect the virus from the cassette into a centrifugal filter unit.
5. Centrifuge at 4,000 x g for 15-30 min at 4 °C.
   NOTE: Rince the AAV with AAV dialysis buffer 2-3 times. Concentrate the AAV until ~200 µL of remaining is on top of the filter.
6. Collect the concentrated AAV from the filter unit. Rinse the filter with another 300 µL of AAV dialysis buffer and transfer it to the concentrated AAV.
7. Filter the virus through a 0.22 µm syringe filter using a 1 mL tuberculin luer slip syringe. To ensure the least amount of volume loss, use the 10 mL syringe to push air through the filter 2-3 times.
8. Store the purified AAV virus at 4 °C temporally for titration.
   NOTE: Titrate the virus, aliquot, and store at -80 °C within 1 week.

9. AAV virus titration

NOTE: TaqMan quantitative polymerase chain reaction (qPCR) was used to titrate purified AAV.

1. Treat AAV vectors with DNase I and incubate at 37 °C for 30 min, then incubate at 95 °C for 10 min.
   NOTE: As an example of 10 µL reaction, 2 µL AAV virus sample, 1 µL DNase, 1 µL DNase buffer, and 6 µL H₂O is used. It can be performed in a thermocycler with PCR tubes.
2. Prepare the primer sets targeting the AAV insert region.
   NOTE: Targets could be the promoter, transgene, and reporter in the AAV construct. The primers are listed in Table 5.
3. Prepare DNA plasmid standards (10, 1, 0.1, 0.01, 0.001, and 0.0001 pg/µL) and a negative control (H₂O).
4. Prepare the qPCR master mix, including primers, according to the manufacturer's instructions.
5. Place the standards and samples in triplicate on a PCR plate. Add the qPCR mix into the standards and samples. Seal the plate and perform the qPCR reaction according to the manufacturer's instructions.
   NOTE: The reaction conditions depend on the materials/reagents and instrument. Fast and conventional PCR cycles are applicable.
6. Calculate AAV virus titer.
   NOTE: The sample concentration in this protocol is 1/10 dilution from the original samples.
7. Aliquot the AAV virus in 1.5 mL tubes and store at 4 °C for up to one month and store at -80 °C for the long term.
   NOTE: Avoid freeze/thaw cycles for the storage. When used for in vivo experiments, do not use thaw over twice aliquots.

10. Quality control of AAV

NOTE: AAV viruses were characterized for purity by SDS-PAGE silver stain using an SDS-PAGE gel and stained using a commercially available staining kit.

1. Denature 3 x 10⁹ vg of AAV virus sample with Laemmli buffer.
   NOTE: Use a reference AAV virus as a control. The AAV6-CMV-GFP reference full capsid is used.
2. Load the denatured AAV on a 4%-20% gradient SDS-PAGE gel and run at 180 V for 50 min.
3. Remove the gel from the electrophoresis plate. Stain the gel with a silver staining kit according to the manufacturer's instructions.
4. Check the AAV capsid proteins VP1, VP2, and VP3.
   NOTE: Pure AAV virus contains only VP1, VP2, and VP3 capsid protein. If not pure, other protein bands would be visible on the gel.
   NOTE: Transmission electron microscopy (TEM) of negative-stained recombinant AAV virions was performed to assess morphological integrity and full/empty ratio.
5. Pick up an EM grid with a tweezer and set down on the bench with the shiny side of the grid facing up.
6. Pipet 5 µL of AAV sample onto the grid and allow it to dry by evaporation.
   NOTE: Dilute the AAV if needed. 10¹² vg/mL is an average titer. It might take 30-60 min to dry the grid.
7. Wash the grid by pipetting, drop by drop, with about 200 µL of H₂O onto the grid.
8. Remove the excess water by slowly placing a chromatography paper vertically next to the grid.
9. Pipet 5 µL of 2% Uranyl Acetate solution onto the grid. Incubate for 5 min and wick off as mentioned above. Allow the grid to dry.
10. Visualize the AAV particles under a transmission electron microscope (at 50,000-fold magnification). Viral capsids with a viral genome will appear as homogeneous white hexagons, while empty capsids will appear as hexagons with a white rim but a dark center.
11. Count randomly at least 100 particles to determine the approximate percentage of full vs. empty AAV particles.