AAV-Production-293T

Protocol 1

1. Culture 10⁷ 293T cells in ten 15cm culture dishes one day prior to transfection.

2. Add 300 ÎŒg of AAV vector plasmid and 1400ÎŒg of packaging plasmid to 10 mL of DMEM.

3. Add GeneExpresso Max to 10 ml DMEM.

4. Add GeneExpresso Max in DMEM to DNA in DMEM, mix and incubate for 20 minutes at room temperature.

5. Add 2 ml transfection mixture to each plate

6. Incubate cells for 37ºC and 5% CO2.

7. Remove transfection media after 15 hours and add fresh culture media.

8. Harvest cells two days after transfection.

9. Pellet cells by centrifugation and resuspend in 15mL of 50mM Tris (pH 8.5) containing 150mM NaCl (or PBS, pH 7.5, 1 mM, MgCl2 and 10 mg/ml DNase I)

10. Lyse cells by three freeze-thaw cycles (dry ice/100% ethanol bath and 37°C water bath).

11. Add benzonase to lysate at a final concentration of 50U/mL and incubated at 37ºC for one hour.

12. Prepare iodixanol gradient in 35mL ultracentrifuge tubes, overlaying (from bottom to top) 5mL of 60% iodixanol, 5mL of 40% iodixanol, 6mL of 25% iodixanol and 9mL of 15% iodixanol.

13. Add cell lysate gradient tube and  seal the tube sealed with rotor cap accessory.

14. Spin at 350000g for two hours.

15. Remov AAV from the clear 40% layer by making an air hole on the top of the tube and removing the layer with a needle.

16. Purify AAV using HiTrap heparin column (or heparin-agarose beads column, Sigma, H-6508)  by FPLC.

17. Make 200 µl aliquots and store in a –80ºC freezer.

Protocol 2

1. Seed 293 cells such that they will be at 80% confluency at the time of transfection.

Change medium 3–5 h prior to transfection.

2. Transfect each 150-mm dish with 7.5 ÎŒg of vector plasmid, 7.5 ÎŒg, pRepCap, and 22.5 ÎŒg pHelper (Ad helper plasmid). Change medium after 15–20 h.

3. Harvest cells at 60–70 h post-transfection by scraping into PBS. Collect cells by centrifugation (1000g for 15 min at 4°C) and resuspend in 15 mL Gradient Buffer.

4. Freeze/thaw three times in dry ice/ethanol bath. Add 50 U/mL Benzonase and incubate for 30 min at 37°C and remove cellular debris by centrifugation at 3700g for 15 min at 4°C.

5. Transfer 14 mL of clarified lysate into a Quick-Seal Ultra-Clear centrifuge tube using a syringe equipped with a spinal needle.

6. Dispense 9.7 mL of 15% iodixanol solution into the tube below the lysate, followed by 6.4 mL of the 25% solution, 5.4 mL of the 40% solution, and 5.4 mL of the 60% solution.

7. Fill with buffer, seal and centrifuge in a Type 50.2 Ti rotor (Beckman) at 278,000g for 1 h 45 min at 18°C.

8. Puncture tube with an 18-gage needle and aspirate 40% iodixanol fraction containing the rAAV.

9. Titer virus and store at 4°C.

Purification of Recombinant Rep Protein

1. Inoculate overnight culture of LB with BL21 cells. Grow at 37°C in the presence of 25 ÎŒg/mL kanamycin and 100 ÎŒg/mL ampicillin.

2. Use overnight culture to inoculate 1 L of LB media and grow cells to an OD600 of 0.8.

3. Induce cells with 0.1 mM IPTG for 1 h at 37°C.

4. Pellet cells at 6000g for 20 min at 4°C, wash in 50 mM NaPO4 monobasic pH 8.1, 1 M NaCl and repellet.

5. Resuspend in 20 mL of column buffer. Freeze/thaw twice in dry-ice/ethanol bath.

6. Add lysozyme to 1 mg/mL and incubate on ice for 30 min.

7. Sonicate for 30 s on ice (output 6 on a Branson Sonifer 250, VWR Scientific).

8. Spin at 14,000g at 4°C for 30 min and collect supernatant.

9. Apply 5 mL of extract to a 2-mL Ni-NTA agarose column (QIAGEN) equilibrated in column buffer with 20% glycerol at a flow rate of 0.5 mL/min.

10. Wash with 5 vol column buffer supplemented with 100 mM imidazole and 20% glycerol.

11. Elute in an ascending linear gradient of 0.1 to 1.0 M imidazole and identify fractions containing Rep68H6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining.

12. Pool peak fractions and dialyze into 25 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM DTT, 0.1% Tween-20, 20% glycerol and store at –80°C.

Large scale production of adeno-associated virus

1. Seed 1.2 × 10^7 293 cells in 150-mm dishes.

2. Transfect 7.5 ÎŒg vector plasmid and 37.5 ÎŒg of the pRepCap-Helper plasmid using GeneExpresso Max

3. After 72 h, crap the cells in the media and spin at 1000g.

4. Suspend the pellet in a minimal amount of PBS (1 mL for each 150-mm dish of cells) and subject to 3–5 freeze-thaw cycles followed by sonication.

5. Add sodium deoxycholate to 1.0% and 0.025% trypsin, incubate the lysate at 37°C for 30 min.

6. Remove cellular debris by centrifugation (2000g).

7. Purify virus by two CsCl ultracentrifugations.

8. Add CsCl to the lysate to a final concentration of 1.41 gm/mL.

9. Transfer the suspension to a Beckman centrifuge tube (cat. no. 344059) and centrifuged at 200,000g, using a SW-41 rotor in a Beckman L7/L8-M ultracentrifuge for 48 h.

10. At the end of the spin, take out the tubes, collect 250 ÎŒL fractionations from the top of the tube. Pool the fractions corresponding to a density of 1.38–1.42 g/mL, and add fresh CsCl solution (1.41 g/mL in PBS), then repeat centrifugation.

11. At the end of the second centrifugation collect and pool fractions corresponding to a density of 1.38–1.42.

12. remove the CsCl by dialysing the pooled virus against four changes of PBS, or using a desalting column. Store the resulting AAV at –80°C until used.

Purification of adeno-associated virus by iodixanol step gradient ultracentrifugation

1. Transfer clarified lysate to a Quick-Seal Ultra-Clear Ultracentrifuge tube using a 50-mL syringe and 19-gauge needle.

2. Using a Pasteur pipet fitted to a pipet-aide, underlay the lysate with iodixanol fractions:

9 mL 15% iodixanol/1 M NaCl
6 mL 25% iodixanol/phenol red
5 mL 40% iodixanol
5 mL 60% iodixanol/phenol red

heat-seal ultracentrifuge tubes.

3. Spin using a Beckman Ultracentrifuge and a Type 60Ti rotor at 60,000 rpm (18°C, 362,477g [max]) for 90 min.

4. Collect the 40% fraction (phenol red in the 25% and 60% iodixanol fractions). The lower 60% fraction is yellow due to the low pH of the iodixanol.

Use a 19-gauge needle and 10-mL syringe to remove the 40% fraction (by carefully puncturing the tube at the 60%/40% interface and withdrawing no more than 4.5 mL of the fraction).

Purification of adeno-associated virus by heparin affinity chromatography

1. Attach a 1-mL HiTrap heparin column to a 30-mL syringe with a concentric luerlok.

2. Equilibrate column with 15 mL 1X PBS-MK

3. Load fraction containing AAV to cloumn using a 5-mL syringe. Collect the effluent and pass it through the same column again.

4. Wash column with 15–20 mL 1X PBS-MK.

5. Elute virus from the column by applying 6–7 mL 1X PBS-MK/1 M NaCl.

6. Dilute eluent with 1X PBS-MK to 15 mL.

8. Concentrate virus by ultrafiltration (to approximately 1 mL). Repeat dilution and spin, then spin to 150–300 ÎŒL.

9. This will yield 5 × 10^11 to 2 × 10^12 viral particles (titer is as high as 1 × 10^13 viral particles/mL).

10. Aliquot the virus and store at –80°C.

Production of recombinant adeno-associated virus by CaCl2 Transfection

1. Split 293T cells into 10 15-cm dishes at a cell density of between 8–9.5 × 10^6 cells/dish, so that the cultures are about 70% confluent the following day.

2. On the day of the transfection, prepare the following transfection mixture in a 50-mL Falcon tube (Nunc) by combining 700 ÎŒg pAAV and ppRepCapH at an equimolar ratio, 1.25 mL 2.5 M CaCl2, and make up to 12.5 mL with ddH2O.

3. Add 12.5 mL 2X HBS (room temperature) at a steady slow rate.

4. Incubate the transfection mixture for 1 min at room temperature, beginning from the addition of the 2X HBS.

5. Transfer the entire 25-mL transfection mix to 200 mL of prewarmed growth medium (37°C/8% CO2)

6. Mix gently by inversion.

7. Remove culture medium from 15-cm dishes

8. Add 22 mL of the transfection mix to each plate.

9. Incubate at 37°C/8% CO2 for 2 to 3 d.

10. Remove ~ 20 ml culture medium, leaving 2–3 mL in each plate.

11.Scrap cells, pool cells and pellet cells by centrifugation at 1000g for 10 min.

12. Resuspend cell pellet in 15-mL cell lysis buffer.

13. Lyse cells by four cycles of freeze/thaw by alternatively placing in ethanol/dry ice and then a 37°C water bath.

14. Add 3 ÎŒL of benzonase to the 15-mL lysate and vortexed briefly, then incubate at 37°C for 1 h.

15. Centrifuge at 6500 rpm (18°C, 5000g) for 20 min to clarify the cell lysate.

16. Store the clarified lysate at –80°C.

Purification of adeno-associated virus using Heparin Column

1. Preequilibrate a heparin column with 10 mL 150 mM NaCl, 20 mM Tris-HCl pH 8.0.

2. Load the 50 mL of aav at a flow rate of approx 2–2.5 mL/min.

3. Wash column with 20 mL 100 mM NaCl, 20 mM Tris-HCl pH 8.0.

4. Wash and elute, collect in 1.5-mL sterile Eppendorf tubes.
1X 1 mL 200 mM NaCl, 20 mM Tris-HCl pH 8.0—discard;
1X 1 mL 300 mM NaCl, 20 mM Tris-HCl pH 8.0—discard;
1X 1 mL 400 mM NaCl, 20 mM Tris-HCl pH 8.0;
2X 1 mL 450 mM NaCl, 20 mM Tris-HCl pH 8.0;
1X 1 mL 500 mM NaCl, 20 mM Tris-HCl pH 8.0.

5. Pool the 400 mM–500 mM NaCl, 20 mM Tris-HCl pH 8.0 fractions (4 mL), and concentrate using a 100K MWCO centrifugal concentrator.

6. Dialyze aav preparation overnight at 4°C against two changes of 1 L PBS containing 1 mM MgCl2 using Pierce Slide-A-Lyzer dialysis cassettes, 3500 MWCO.

7. Filter sterilize using a 0.2-ÎŒm 13-mm low-protein binding Acrodisc filter.

8. Aliquote and store at –80°C.

Production of recombinant adeno-associated virus by CaCl2 Transfection: Protocol 3

1. Day 1: 24 h prior to transfection, seed 15-cm tissue culture plates with 2.0–2.2 × 10^7 293 cells per plate in 25 mL DMEM. The plates should be approx 70% confluent at the time of transfection.

2. Day 2: 2–3 h before the transfection, remove the culture media and replace with 25 mL IMDM per plate, prewarmed to 37°C.

3. Allow the 2X HeBS buffer, 2.5 M CaCl2 and distilled H2O to equilibrate to room temperature.

4. In Nunc 50-mL tubes, prepare the transfection mix. For each 15-cm dish, use 66 ÎŒg helper plasmid (pRepCapHelper), 11 ÎŒg AAV plasmid, 330 ÎŒL 2.5 M CaCl2 and make up to a final volume of 2.75 mL with distilled water.

5. Filter the transfection mix through a 0.2-ÎŒm Acrodisc filter to sterilize and remove particles that may bind to the calcium phosphate precipitate. Dispense the solution into 2.5-mL aliquots in sterile disposable polystyrene tubes.

6. Take an aliquot of transfection mix and while vortexing the solution vigorously, quickly add 2.5 mL of 2X HeBs buffer with a pipet gun and keep vortexing for a further 5–10 s. Leave the solution to stand for a further 50 s (total of approx 1 min for precipitate to form).

7. Take a plate out of the incubator and quickly, but gently, add the 5-mL transfection solution dropwise in a circular motion around the plate, then swirl gently to mix, and return plate to the incubator.

8. 8–15 h after transfection, aspirate the medium and replace with 25 mL of prewarmed DMEM.

9. 48–60 h after transfection aspirate the media from the cells and discard.

10. Add 25 mL prewarmed PBS to the plate, aspirate, and discard.

11. Add 25 mL of PBS to each plate and detach cells using a cell scraper. Transfer the contents to 50-mL Nunc tissue culture tubes.

12. Pellet cells at 200g for approx 5 min.

13. Remove supernatant and resuspend cell pellets in 150 mM NaCl, 20 mM Tris-HCl pH 8.0. Volume to use is 10 mL per plate.

14. Pool the cell suspension and split into two 25-mL aliquots in 50-mL Nunc tubes.

15. Add sodium deoxycholate to a final concentration of 0.5% (1.25 mL of a 10% stock solution to each tube containing 25 mL).

16. Add Benzonase to a final concentration of 50 U/mL (4.25 ÎŒL of 300 U/ÎŒL to each tube).

17. Incubate in a 37°C water bath for 30–40 min, before removing cell debris by centrifugation at 3000g × 15 min, 4°C.

18. Transfer the supernatant into another Nunc tube and heat in a 56°C water bath for 15 min.

19. Freeze supernatant in a dry ice/ethanol bath.

20. Thaw the supernatant and remove any cell debris by centrifugation at 3000g × 15 min, 4°C.

21. Pool the two 25-mL aliquots to give a total of 50 mL from the five plates. Filter through a 32-mm 0.45-ÎŒm Acrodisc syringe filter to remove any particulate matter

Titration of Recombinant AAV real time PCR.

Extraction of Viral DNA

1. Dilute 2 ÎŒL of the virus stock in 80.5 ÎŒL PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.01% gelatin).
2. Add 350 U (17.5 ÎŒL) of DNaseI at 37°C for 30 min.
3. Inactivate DNase at 70°C for 10 min.
4. Add 10 ÎŒg of proteinase K and incubate 1 h at 50°C.
5. Inactivate proteinase K at 95°C for 20 min.
7. Store virus DNA at –20°C.

Dilution Series of the aav DNA

Transfer 22 ÎŒL vector DNA into a sterile 0.5-mL tube (50X).
1. Dilute 2 ÎŒL of the stock solution in step 1 with 20 ÎŒL sterile water (500X).
2. Dilute 2 ÎŒL of the stock solution in step 2 with 20 ÎŒL sterile water (5000X).
3. Dilute 2 ÎŒL of the stock solution in step 3 with 20 ÎŒL sterile water (50,000X).
4. Dilute 2 ÎŒL of the stock solution in step 4 with 20 ÎŒL sterile water (500,000X) and discard 2 ÎŒL of the final dilution.
5. Add 80 ÎŒL sterile water to each tube and use 5 ÎŒL per PCR.

PCR

Set up standard samples, diluted virus DNA and nontemplate control in duplicates. Add 5 ÎŒL diluted virus DNA, standard, or water per tube, respectively. Then add the following to each tube, so that the total volume for each reaction is 25 ÎŒL.

WPRE-F (10 uM) 0.5 ÎŒL;
WPRE-R (10 ÎŒM) 0.5 ÎŒL;
5X SYBR Green Mix 12.5 ÎŒL;
H2O 6.5 ÎŒL

Cell Biolab Protocol

1. One day before transfection, plate sufficient 293 cells or 293AAV cells (Cat. # AAV-100) to
achieve 70-80% confluence on the day of transfection.
2. Cotransfect cells with pAAV Expression vector, pAAV-RC and pHelper.
Notes:
• We recommend the ratio of vectors at 1:1:1 (pAAV Expression Vector:pAAV-RC:pHelper).
• Calcium Phosphate transfection method is preferred for AAV production. For lipid-based
transfection reagents, we only suggest FuGENE® 6 (Roche Applied Science) or
LipofectamineTM LTX (Invitrogen).
3. 48-72 hours after transfection, add 0.5 M EDTA to a final of 10 mM to the plate 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.
Notes:
• As viral production proceeds, some of the cells will round up and detach from the plate, and
can be seen as floating in the medium.
• Viruses are present in both intact cells and the growth medium. For more concentrated virus
stock, we only recommend proceeding with cell pellet.
4. Centrifuge the cell suspension at 1000 RPM for 5 min. Remove the supernatant and resuspend the
cell pellet in desired amount of DMEM or sterile PBS.

Freeze and thaw the cell suspension four times by placing it alternately in a dry ice/ethanol bath
and a water bath of 37oC. Remove cell debris by centrifugation at 10,000 g for 10 min and collect
the supernatant as AAV crude lysate.
6. AAV crude lysate can be used directly or purified/concentrated if needed. For long term storage,
store supernatant at -80oC in aliquots.

The quality of rAAV vector preparations is a key element in obtaining reliable and reproducible data.
Purification of rAAV from crude cell lysate is usually required before transduction of your target cells.
rAAV is usually quantified by genome copy (GC) number. These genome-containing particles are
functional vectors that infect target cells. Besides these "full" AAV, empty viral particles are also
produced. Measurement of GC rather than total particle number is thus more relevant.
1. Concentration and purification of your rAAV: Recombinant AAV vector can be purified by
CsCl gradient ultracentrifugation, iodixanol discontinuous gradient ultracentrifugation, and high-
performance liquid chromatography (HPLC). For AAV-2, we recommend using Cell Biolabs’
ViraBindTM AAV Purification Kit (Catalog # VPK-140).
2. Measure titer of your rAAV:
a. Genome Copy (GC) Number: This is an important step to ensure consistent viral transduction
into your host cell. However, QPCR or dot blot of viral DNA can take as much as 1-4 days to
complete. An ELISA method has been developed by using antibody that only reacts with AAV
intact particles; however, this method measures all AAV particles including the ones lacking
genomic DNA. Cell Biolabs’ QuickTiterTM AAV Quantitation Kit (Catalog # VPK-145) does
not involve cell infection; instead it specifically measures the viral nucleic acid content of
purified viruses or unpurified viral supernatant sample. The entire procedure takes about 4
hours for unpurified supernatant or about 30 minutes for purified AAV.
b. Infectious Titer: For AAV vector containing reporter, the rAAV infectious titer can be
determined using either green fluorescent protein (GFP) or LacZ as the reporter gene. For
rAAV-LacZ, each blue cell after X-Gal staining represents one infectious unit (IU). For rAAV-
GFP, each green cell under fluorescence microscopy represents one IU.
3. Use transduction reagents to increase infection efficiency: The AAV transduction process
includes viral binding and entry, intracellular trafficking, nuclear transport, and viral second strand
DNA synthesis. The viral second strand DNA synthesis has been shown to be the rate limiting
step, which leads to inefficient transduction by AAV vectors. Cell Biolabs’ ViraDuctinTM AAV
Transduction Kit (Catalog # AAV-200) is designed to increase transduction efficiencies by AAV
on both dividing and non-dividing cells.