Experiment Summary

Recombinant adeno-associated viruses (rAAVs) are valuable viral vectors for in vivo gene transfer, also having significant ex vivo therapeutic potential. Continued efforts have focused on various gene therapy applications, capsid engineering, and scalable manufacturing processes. Adherent cells are commonly used for virus production in most basic science laboratories because of their efficiency and cost. Although suspension cells are easier to handle and scale up compared to adherent cells, their use in virus production is hampered by poor transfection efficiency. In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent. The established protocol allows AAV production from transfection to quality analysis of purified AAV within two weeks. Typical vector yields for the described suspension system followed by iodixanol purification range from a total of 1 × 10¹³ to 1.5 × 10¹³ vg (vector genome) using 90 mL of cell suspension vs. 1 × 10¹³ to 2 × 10¹³ vg using a regular adherent cell protocol (10 × 15 cm dishes).

Materials and Reagents

1. HEK293T adapted for serum-free media

2. pAdΔF6 helper plasmid

3. pAAV2/9 Rep/Cap plasmid

4. AV0-EF1-N-cG (control AAV transfer plasmid) or AV0-EF1-N-tdT

5. DNase I

6. RNase A

7. Forward qPCR primer such as WPRE or other vector specific primer

8. Reverse qPCR primer such as WPRE or other vector specific primer

9. BalanCD HEK293 media liquid or powder

10. GlutaMAX

11. 100× Pluronic F-68

12. VirusGEN AAV Transfection kit

13. 1 M Tris-HCl pH 8.0, NaCl, MgCl₂, KCl

14. 1 M DTT

15. Glycerol

16. Sodium deoxycholate

17. HEPES

18. Sarcosyl

19. EDTA

20. PEG8000

21. OptiPrep Density Gradient

22. Phenol red

23. DPBS

24. HyPure molecular biology grade water

25. SYBR Green SuperMix

26. Sodium bicarbonate

27. Trypan blue

A. Solutions

1. Complete BalanCD HEK293 media (c-BalanCD HEK293)

2. DNase I solution

3. RNase A solution

4. 2 M MgCl₂, 5 M NaCl, 2.5 M NaCl

5. 40% PEG8000/2.5 M NaCl

6. TMN

7. HBS

8. 5% sodium deoxycholate

9. PBS-MK

10. PBS-NMK

11. DPBS/Pluronic F-68

12. BalanCD HEK293 media-powder reconstituted

Procedure

A. Defrosting and cell maintenance

1. Defrost cells into 30 mL of warm c-BalanCD HEK293 media into a 125 mL baffled Erlenmeyer flask.

2. Place the flask onto the shaker platform in the 37 °C and 5% CO₂ incubator at 120 rpm.

3. Count cell density every other day and dilute cells with c-BalanCD HEK 293 media as needed to maintain the cell density between 3 × 10⁵ live cells/mL and 3 × 10⁶ live cells/mL.

B. noculate flask (Day 1)

1. Warm c-BalanCD HEK293 media at 37 °C in a water bath.

2. Inoculate a 250 mL baffled Erlenmeyer flask with 1 × 10⁶ live cells/mL in a total of 90 mL of c-BalanCD HEK293 media.

3. Place the 250 mL baffled Erlenmeyer flask on the shaker platform in the 37 °C and 5% CO₂ incubator at 120 rpm.

C. Transfection (Day 2)

1. Ensure that the cell density of the previously inoculated 250 mL baffled Erlenmeyer flask reaches 1.0-2 × 10⁶ live cells/mL (1 day after inoculation). Follow the method in step A3h to determine cell counts.

2. Transfer 0.5 mL of the cell suspension to a 24-well plate as a negative control.

3. Add 9 mL of complex formation solution and enhancer (CFSE) from the VirusGEN AAV Transfection kit .to a 15 mL centrifuge tube.

4. Add the appropriate quantity of plasmid DNA to the CFSE in the 15 mL centrifuge tube and gently reflux to combine for 10 s. Table 1 shows the DNA quantities needed for each component of the transfection mixture.

Table 1. DNA quantities

5. Add 270 µL of TransIt-VirusGen Transfection Reagent (3 µL per 1 mL of cells) to the CFSE/DNA mixture and gently reflux to mix well.

6. Let the transfection mixture incubate at room temperature for 30 min.

7. After the incubation period is over, gently reflux the transfection mixture well and add directly to the 250 mL flask while manually swirling the flask.

8. Put the 250 mL baffled Erlenmeyer flask back on the shaker platform in the 37 °C and 5% CO2 incubator at 120 rpm.

D. Harvesting after 72 h (Day 5)

1. (Optional) Transfer 0.5 mL of cell suspension to a 24-well plate and observe fluorescent protein expression if it exists. See Figure 1 for what a typical transfection efficiency may look for a transgene plasmid that had a fluorescent marker.

Figure 1. Transfection efficiency gauged by fluorescence.

2. Transfer the entire contents of the 250 mL baffled Erlenmeyer flask (this should be approximately 100 mL of cell/media suspension) to a newly labeled 250 mL centrifuge tube.

3. Wash the flask with 24 mL of DPBS and combine with cell/media suspension.

4. Centrifuge cell/media suspension at 335× g for 10 min at room temperature. See Figure 2 for what this may look like.

Figure 2. Centrifuged cell pellet and supernatant 72 h after transfection with AV0-EF1-N-tdT

E. Digestion (Day 5)

1. Pour supernatant carefully into a newly labeled 250 mL bottle, being careful not to disturb the cell pellet.

2. For the supernatant, complete the following steps:

a) To the new 250 mL bottle with the supernatant in it, add 150 µL of DNase I solution, 150 µL of RNase A solution, and 1 mL of 2 M MgCl₂.

b) Invert the centrifuge tube to mix and incubate for 1 h in the 37 °C and 5% CO₂ incubator.

c) After 1 h, add 25 mL of 40% PEG 8000/2.5 M NaCl for every 100 mL of digested supernatant.

d) Invert the bottle at least 20 times to mix and store at 4 °C until purification.

3. For the cell pellet, complete the following steps:

a) Resuspend the cell pellet in 8 mL of TMN.

b) Loosen the pellet by tapping the bottom of the centrifuge tube against the metal surface of the cell culture hood.

c) Resuspend the cell pellet in the TMN using a pipette controller and transfer the mixture to a 15 mL centrifuge tube.

d) Add 10% by volume of 5% sodium deoxycholate and rock for 30 min at room temperature.

e) Add 100 µL of 2 M MgCl₂, 150 µL of DNase I solution, and 150 µL of RNase A solution.

f) Mix and incubate for 1 h in a 37 °C water bath. Vortex the tube every 15 min.

g) Freeze at -80 °C until purification.

F. Preparation for purification (Day 6)

1. When ready to purify, complete the following steps for the supernatant:

a) Remove the 250 mL centrifuge tube from the 4 °C refrigerator and centrifuge it at 410× g for 30 min at 4 °C.

b) Aspirate the supernatant carefully, ensuring to leave the pellet intact at the bottom.

c) Add 2.5 mL of HBS to the pellet.

d) Loosen the pellet by tapping the bottom of the centrifuge tube against the metal surface of the cell culture hood.

e) Resuspend the pellet in the HBS using a pipette controller and transfer the mixture to a 15 mL centrifuge tube.

f) Vortex the HBS/pellet homogenate well.

g) Place the 15 mL centrifuge tube on a rocker and rock until the pellet dissolves completely in the HBS. This will take at least 2 h, but likely longer.

2. When ready to purify, complete the following steps for the cell pellet:

a) Remove the cell pellet suspension from the -80 °C freezer and thaw completely in a 37 °C water bath.

b) Once thawed, add 100 µL of DNase I solution and 100 µL of RNase A solution.

c) Incubate the suspension in a 37 °C water bath for 1 h.

d) Centrifuge the cell pellet suspension at 410× g for 10 min at 4 °C.

3. Carefully combine the cleared cell lysate solution with the dissolved HBS/pellet mixture. Be careful not to touch any of the cell debris collected at the bottom of the 15 mL centrifuge tube in which the cell pellet solution was originally in.

4. Place this solution into the 4 °C refrigerator momentarily until the purification gradient is made, as described in the next step.

G. Purification (Day 6)

1. Prepare four solutions for the different densities that will be used to create the OptiPrep purification density gradient. Table 2 describes the quantities of OptiPrep, PBS-MK or PBS-NMK, and phenol red needed for these solutions.

Table 2. OptiPrep gradient components

2. Into a sterile thin-wall polypropylene centrifuge tube, start by adding 7 mL of 15% OptiPrep to the bottom of the tube.

3. Carefully underlay 5 mL of 25% OptiPrep beneath the 15% OptiPrep layer.

4. Carefully underlay 5 mL of 40% OptiPrep beneath the 25% OptiPrep layer. See Figure 3 for how this underlay should look.

Figure 3. OptiPrep underlay.

5. Finally, carefully underlay 4 mL of 60% OptiPrep beneath the 40% OptiPrep layer. See Figure 4 for what a successfully created gradient should look like.

Figure 4. Complete gradient.

6. Outline the top and bottom interfaces of the 40% OptiPrep layer with a fine-tipped marker for a reference point during extraction.

7. Remove the cleared cell pellet suspension solution combined with the HBS/pellet mixture from the 4 °C refrigerator.

8. Centrifuge the combined solutions' 15 mL centrifuge tube at 5,000× g for 10 min at 4 °C.

9. Carefully overlay the combined solution over the OptiPrep gradient. Be careful not to pipette any cellular debris that may have accumulated at the bottom of the 15 mL centrifuge tube. See Figure 5 for what the gradient should look like with the combined solution overlayed on top.

Figure 5. Gradient with crude virus before ultracentrifugation.

10. Transfer the thin-wall polypropylene centrifuge tube to the SW32Ti swinging-bucket rotor in the ultracentrifuge.

11. Centrifuge at 160,713× g for 15 h at 20 °C (overnight centrifugation).

H. Band extraction (Day 7)

1. After the ultracentrifuge has stopped, remove the thin-wall polypropylene centrifuge tube from the rotor.

2. Remove the thin-wall polypropylene centrifuge tube from the adaptor and place it into a holder. See Figure 6 for what the gradient should look like after ultracentrifugation.

Figure 6. Gradient after ultracentrifugation.

3. Prepare a 50 mL centrifuge tube in a rack.

4. Attach a 20 G sterile hypodermic needle to a 5 mL sterile syringe.

5. Use an alcohol wipe to disinfect the puncturing location (the bottom interface of the 40% OptiPrep layer that you previously marked).

6. Apply even pressure and puncture the thin-wall polypropylene centrifuge tube at the previously marked bottom interface of the 40% OptiPrep layer. See Figure 7 for guidance on where to puncture the tube.

Figure 7. Band extraction.

7. Extract 4–4.5 mL of the OptiPrep solution, being very careful not to accidentally extract any debris that may have sedimented at a different density in the gradient.

8. Empty the contents of the syringe into the pre-prepared 50 mL centrifuge tube. Be sure to squeeze out as much of the extracted solution as possible before disposing of the needle and syringe in the appropriate sharps container.

9. Add 45 mL of DPBS/0.001% Pluronic F-68 up to the 50 mL mark of the 50 mL centrifuge tube and place at 4 °C.

I. Concentration of virus (Day 7)

1. Add 15 mL of DPBS/0.001% Pluronic F-68 to an Amicon centrifugal filter unit and let it equilibrate for 15 min at room temperature.

2. Centrifuge the Amicon filter at 2,096× g for 2 min at 20 °C. Most, but not all, of the DPBS/0.001% Pluronic F-68 should have flowed through to the collection reservoir.

3. Empty the collection reservoir into a waste bucket.

4. Add the diluted AAV in the 50 mL centrifuge tube to the Amicon filter and centrifuge the Amicon filter at 2,096× g for 2 min at 20 °C.

5. Empty the flowthrough from the collection reservoir before adding any more diluted AAV.

6. Repeat the previous steps I3 and I4. Add diluted AAV to the Amicon filter. Centrifuge at 2,096× g for 2 min at 20 °C until the entirety of the diluted sample has been applied to the Amicon filter.

7. When the diluted AAV is exhausted, add 30 mL of DPBS/0.001% Pluronic F-68 to the 50 mL centrifuge tube to rinse it out.

8. Add the rinse solution from the 50 mL centrifuge tube to the Amicon filter and centrifuge at 2,096× g for 2 min at 20 °C until the rinse solution is exhausted as well.

9. Centrifuge at 2,096× g for 1 min at 20 °C as many times as needed until the liquid level in the Amicon tube reaches 200 µL.

10. Transfer the entire 200 µL of purified AAV from the Amicon filter to another tube and store at 4 °C for up to six months. For longer storage, aliquot the purified AAV into smaller quantities and store at -80 °C.

J. QPCR for genome titer (Day 8)

Data Analysis

After the qPCR cycle has been run, you should analyze the data. Export the text data and calculate the physical titer: Titer (vg/mL) = [1 × 1012 × (Concentration in ng/µL from qPCR software) × (dilution factor)]/3.222 ng.