Novel Approaches for Production of Recombinant Adeno-Associated Virus

Novel Approaches for Production of Recombinant Adeno-Associated Virus

This article describes how to produce a recombinant adeno-associated virus (rAAV) stock of high purity. The adeno-associated virus (AAV) is a human parvovirus that was first engineered as a recombinant viral vector for gene delivery by Hermonat and Muzyczka in 1984. The unique biology and life cycle of rAAV make it a popular choice as a gene delivery system as it satisfies the main criteria for successful gene vectors. These criteria include but are not limited to (1) efficient transduction of the target cell; (2) stable and long-term expression of the transgene of interest, especially with the use of promoters and enhancers that are not inactivated in the transduced cell; and (3) a lack of stimulation of a cytotoxic immune response to the vector or transduced cell, resulting in a very good safety profile for clinical application. Wild-type AAV has not previously been associated with disease in healthy adult humans and is classified as a risk group 1 agent under the NIH's recombinant DNA guidelines (rev. 04/02). Recombinant AAV vectors retain only the inverted terminal repeat (ITRs) sequences from the wild-type AAV genome, with 96% of the DNA genome removed. This includes all viral coding genes. Normally recombinant AAV is considered nonpathogenic, noninfectious, and nonhazardous.

However, the incorporation of oncogenes or toxin encoding genes into the vector genome may alter this status. Therefore, laboratory facilities used to produce rAAV may be required by local institutions to operate in accordance with biosafety level 2 guidelines, despite rAAV not being a risk group 2 agent in typical circumstances. Wild-type AAV requires helper functions provided in trans by a helper virus such as adenovirus or herpes virus for AAV replication. Early generation rAAV preparations were produced using a helper adenovirus that was then almost completely eliminated in the purification process. More recently, trace levels of helper virus in rAAV stocks have been shown to elicit a cellular immune response to the AAVtransduced tissue (Monohan et al., 1998). Soon after this observation, efforts were made to improve the procedure for generating rAAV vectors, with our laboratory developing a packaging procedure that uses nonoverlapping plasmid constructs to produce rAAV vectors free of contamination by wild-type AAV or helper adenovirus (Xiao et al., 1998). All AAV vectors utilize a plasmid substrate carrying the viral ITR sequences flanking the therapeutic gene of interest. For efficient packaging, the rAAV insert size must be ~4.6kb or smaller, consistent with wild-type genome size ~4.7kb. The AAV plasmid is then cotransfected into human embryonic kidney (HEK) 293 cells, along with a plasmid(s) that provides AAV and adenovirus helper functions. HEK 293 cells contain an adenovirus 5 EIA gene integrated into the genome that activates the AAV Rep and Cap, as well as other essential Ad genes required for productive AAV infection. In this setting, only the gene insert along with the flanking ITRs is then packaged into rAAV virions. Major advances in AAV production have been directly related to better understanding the unique biology of this virus. For example, Summerford and Samulski (1998) identified the primary receptor for AAV type 2 as heparin sulfate proteoglycan. As a result, a novel purification procedure using affinity chromatography was developed to generate virus stocks with a very high level of purity. In addition to the affinity chromatography step, this protocol also uses an iodixanol gradient in place of the cesium chloride step used in earlier protocols to significantly shorten the highspeed centrifugation step and improve the quality of the vector preparations. The aim of this article is to discuss methods for quantifying the purified vectors using the most current approaches that are reproducible from laboratory to laboratory.

A. Cell Culture
Human 293 cells are from American Type Culture Collection (ATCC, Rockville, MD; CRL 1573).

Phosphate-buffered saline (PBS)(No. D-5837; Sigma-Aldrich)

Dulbecco's modified Eagle's medium (DMEM) (No. D- 6429; Sigma-Aldrich)

Penicillin-streptomycin (No. 15140-122) (Gibco-BRL Life Technologies)

Fetal bovine Serum (No. F-2442) (Sigma-Aldrich)

Trypsin-EDTA (No. T-4049) (Sigma-Aldrich)

Falcon integrid tissue culture dish (No. 08-772-6) (Fisher-Scientific)

Light microscope

B. Plasmids
Plasmid with transgene of interest

Plasmid pXX2, the AAV helper plasmid (Samulski laboratory); map and sequence are available on the internet at

Plasmid pXX6, the adenoviral helper plasmid (Samulski laboratory); map and sequence are available at the just-listed web address

C. Production of Adenovirus-Free Recombinant Virus
Monolayers of 293 cells at approximately 80% confluency

Corning 50-ml concical centrifuge polystyrene tubes (No. 05-538-55A, Fisher)

2X HeBs (HEPES-buffered saline): Mix 16.4 g of NaCl, 11.9g of HEPES, and 0.21 g of Na2HPO4 (pH 7.05). Adjust to 1 liter and filter sterilize.

Nalgene nitrocellulose filter sterilization unit (0.45 µm) (1000 ml, No. 09-761-40; Fisher)

2.5 M CaCl2

Filter-sterilized ddH2O

Restriction enzymes Xbal and HindIII.

Sure bacteria (Stratagene)

D. Purification of Recombinant Virus
Sorvall RT 6000B and Sorvall GS3 rotor

Beckman Ultracentrifuge and SW-41 and Ti-70

Ultrasonic processor (Cole-Palmer) with 1/8-in diameter probe (processor No. U-04711-30; microtip No. U-04710-46; Cole Palmer)

Pump Pro (Watson-Marlow) (No. 14-283-13; Fisher)

Ethanol/dry ice bath

Opti-seal tubes (No. 361625; Beckman)

Corning 50-ml conical centrifuge tubes

PBS-MK: Mix 50ml of 10X PBS, 0.5 ml of 1M MgCl2, and 0.5 ml of 2.5 M KCl and adjust to a final volume of 0.5 liter with ddH2O.

Ultraclear tubes (12.5 and 32.4ml) for SW-41 rotor (Beckman)

Heparin sepharose column (1 ml) Hi Trap No. 17-0407: Amersham Pharmacia Biotech

Optiprep (No. 103-0061) (Gibco-BRL Life Technologies)

Opti-mem 1 (No. 31985-013) (Gibco-BRL Life Technologies)

Sodium deoxycholate (No. D-5670; Sigma)

Benzonase (No. E-8263; Sigma)

AKTA FPLC (Amersham-Pharmacia Biotech)

15% iodixanol with 1M NaCl: Mix 5ml of 10X PBS, 0.05 ml of 1 M MgCl2, 0.05 ml of 2.5 M KCl, 10 ml of 5M NaCl, 0.075 ml 0.5% stock phenol red, and 12.5 ml of Optiprep. Adjust to a final volume of 50ml with ddH2O and filter sterilize.

25% iodixanol: Mix 5ml of 10X PBS, 0.05ml of 1M MgCl2, 0.05ml of 2.5M KCl, 0.1ml of 0.5% stock phenol red, and 20ml of Opti-prep. Adjust to a final volume of 50ml with ddH2O and filter sterilize.

40% iodixanol: Mix 5ml of 10X PBS, 0.05ml of 1M MgCl2, 0.05 ml of 2.5M KCl, 33.3 ml of Opti-prep. Adjust to a final volume of 50ml with ddH2O and filter sterilize.

60% iodixanol: Mix 0.05 ml of 1M MgCl2, 0.05ml of 2.5 M KCl, and 0.025 ml of 0.05% stock phenol red in 50ml of Opti-prep. Filter sterilize.

Phenol red (Gibco-BRL Life Technologies)

Slide-A-Lyzer 10,000 MWCO (No. 66451; Pierce)

Syringes (5 ml)

Needles (18 gauge)

Rubber policeman wings (no. 14-110; Fisher)

E. Dot Blot Assay
DNasel digestion mixture: 10mM Tris-HCl, pH 7.5, 10mM MgCl2, 2mM CaCl2, 50U/ml DNase 1

Proteinase K digestion mixture: 1M NaCl, 1% (w/v) Sarkosyl, 200µg/ml of proteinase K

Whatman 3MM paper

Dot blot apparatus

Gene-Screen Plus membrane (New England Nuclear)

Random primer labeling kit (Roche Biomedical)

32p-dCTP (Amersham Pharmacia Biotech)

Church buffer: Mix 5 g of bovine serum albumin, 1 ml of 0.5M EDTA, 33.5 g of Na2HPO4· 7H2O, 1 ml of 85% H3PO4, 35g of sodium dodecyl sulfate (SDS). Adjust to a final volume of 0.5 liter with ddH2O. Heat at 65°C to dissolve. Store on the laboratory bench indefinitely.

Hybridization low-stringency wash solution: 2X saline sodium citrate (SSC), 2X 0.1% (w/v) SDS

Hybridization medium-stringency wash solution: 0.5X SSC, 1X 0.1% (w/v) SDS

Hybridization high-stringency wash solution: 0.1X SSC, 0.5X 0.1% (w/v) SDS

2X SSC: Mix 17.5g of NaCl and 8.8g of trisodium citrate ·2 H2O. Adjust the final volume to 1 liter and adjust the pH to 7.0.

Hybridization bottles (Gibco-BRL Life Technologies)

Phosphoimager or scintillation counter

A. Ad-Free Production of Recombinant Virus

1. Construction of rAAV Plasmid Vector
  1. Modify the plasmid psub201 by digestion with enzymes XbaI and HindIII to remove the Rep and Cap genes. This should produce a fragment less than 4.6 kb and should contain the AAV ITRs.
  2. Insert the foreign gene cassette made of the transgene and its promoter between the XbaI sites.

2. Transfection of 293 cells
Note: Warm up 2X HeBS buffer, 2.5 M CaCl, and filtered distilled water to room temperature.
  1. Seed 293 cells 24 h before transfection at 22 × 106 cells per 15-cm dish in complete DMEM. Generally a total of 20 dishes are transfected for the viral preparation procedure.
  2. Three hours before the transfection, aspirate media and replace with 25ml prewarmed complete 10% FBS/DMEM.
  3. Prepare transfection mixture. Each 15-cm dish is transfected with 25 ml of preformed DNA-CaPO4 precipitate. A total of 37.5 µg DNA per dish will give the optimal precipitate. This should include 7.5 µg vector plasmid, 7.5 µg helper plasmid (i.e., XX2), and 22.5 µg of Ad plasmid (i.e., XX6-80). The precipitates should be formed in reactions of 40-ml. A 40-ml reaction will transfect 20 × 15-cm dishes.
  4. Aliquot DNA mixture into a sterile 50-ml Corning conical tube. Bring volume to 18ml with sterile ddH2O. Add 2ml of 2.5M CaCl2. Aliquot 20ml of 2X HeBS to a separate 50-ml Corning conical tube.
  5. Add the DNA mixture by pipette dropwise to the tube containing the 2X HeBS. Gently mix by inversion. Allow 3-5 min for precipitate to form.
  6. Once precipitate has formed, quickly but gently add 2ml of transfection solution dropwise in a circular motion around the plate. Swirl gently to mix.
  7. Sixteen hours posttransfection, aspirate the media of each plate and replace with 25 ml of complete 2% FBS/DMEM.1

3. Cell Harvesting
Note: Harvesting should occur within 64 h but not less than 48 h of transfection.
  1. Use a rubber policeman to detach cells from dishes using a scraping motion. Pipette the entire mixture into two sterile 500-ml centrifuge bottles (maximum 250ml per bottle). Spin at 5000rpm for 10rain. Pour off the supernatant. (Note: Cell pellets may be frozen in a -20°C freezer overnight if necessary.)

4. Purification of Recombinant Virus
  1. Resuspend the cell pellet in 1X PBS in a sterile 50- ml conical tube.
  2. Add 0.35ml of stock 10% DOC (sodium deoxycholate). Final concentration should be 0.5%.
  3. Add 1.4µl of stock 250µg/µl benzonase. Final concentration should be 50µg/µl.
  4. Incubate at 37°C for 30min.
  5. Spin for 30rain at 6500rpm (8400g). Transfer the supernatant to a sterile 50-ml conical tube.
  6. Freeze/thaw the cell suspension using a dry iceethanol bath and a 37°C H2O bath.
  7. Repeat the freeze/thaw step two more times.
  8. Spin for 20 min at 6500 rpm. Decant the supernatant to a new tube. (Note: Sample may be stored in -80°C freezer at this point).

5. Purification Using an Iodixanol (Optiprep) Gradient
  1. Dissolve the pellet into PBS-MK. Place half (7.5ml) of the resuspended pellet into each of two Optiseal tubes. Using the Pump Pro, create a step gradient by underlying the sample with 6 ml of 15% iodixanol, 6 ml of 25% iodixanol, 7 ml of 40% iodixanol, and 5ml of 60% iodixanol. Carefully remove the tubing without disturbing the gradient layers. (Note: A syringe and small diameter tubing may also be used to layer the gradients.)
  2. Balance the tubes and fill them completely by slowly adding 1X PBS dropwise to the tube to the uppermost layer. Insert a plug and centrifuge at 4°C for 1 h using a Beckman Ti-70 rotor at 70,000rpm (350,000g).
  3. Carefully remove the Optiseal tubes from the rotor. In a viral hood, remove the plug from the top of the tube. Use a 5-ml syringe with an 18-gauge needle to puncture the tube at the 40-60% iodixanol interface. Remove 75% of the 40% iodixanol layer.

6. Purification Using a Heparin Column
Note: A viral preparation made from twenty 15-cm dishes of 293 cells can be purified on a 1-ml column with a single injection.
  1. Sterilize the lines of the AKTA FPLC with 0.5M NaOH and 1X PBS.
  2. Run the "pump wash" program.
  3. Set up the heparin column and check it for leaks.
  4. Inject the sample and autozero the UV.
  5. Run the 1-ml heparin column program.
  6. Collect 0.5-ml fractions.
  7. Sterilize the lines again using 0.5M NaOH and 1X PBS.
  8. Discard the column.
  9. Test the fractions for the presence of virus using the dot blot hybridization assay and combine the fractions with the highest concentration of virus.

7. Delivery of Recombinant Virus in Vitro
a. Determination of rAAV titer by Dot Blot Assay
  1. Place 5µl of each fraction collected from the heparin sulfate column into a well of a 96-well microtiter plate. Assay duplicate samples of each fraction.
  2. Add 50µl of DNase 1 digestion mixture and incubate for 1 h at 37°C. This treatment digests any viral DNA that has not been packaged into capsids.
  3. After 1 h, stop the digestion by adding 10µl of 0.1 M EDTA to each reaction. Mix well.
  4. Add 60µl of proteinase K digestion mixture to each sample to release the viral DNA from the capsid. Incubate for 30rain at 50°C.
  5. To create a set of DNA hybridization standards, use plasmid DNA that was used for the transfection. Linearize the plasmid and do serial dilutions in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. A volume of 25 µl is convenient for each standard in wells of a 96-well microtiter plate. A suitable standard working range is 500 ng to 10 fg.
  6. Denature the samples and control DNA by adding 100µl of 0.5M NaOH to each.
  7. Equilibrate a nylon membrane and one piece of Whatman blotting paper in 0.4M Tris-HCl (pH 7.5) and place them between the upper and lower blocks of a dot blot manifold apparatus; membrane should be on top of the Whatman paper.
  8. Add the denatured DNA from the 96-well microtiter plate to wells of the dot blot manifold apparatus in the absence of a vacuum. After all the DNA has been transferred into the manifold, apply a vacuum for 3-5 min.
  9. Radiolabel a transgene cassette-specific probe. (Note: The probe should not contain plasmid backbone or ITR sequences.)
  10. In a hybridization bottle, prehybridize the nylon membrane with 5 ml of Church buffer for 5 min at 65°C. Discard the prehybridization Church buffer and replace with 5 ml of fresh Church buffer. Place at 65°C.
  11. Boil the 32p-dCTP-radiolabeled probe for 5 min, place on ice, and add to the hybridization bottle containing the dot blot. Hybridize overnight at 65°C.
  12. Remove the hybridization solution and add 10 ml of low-stringency wash solution. Wash for 10 min at 65°C. Repeat the wash with 10ml of fresh solution.
  13. Wash the dot blot for 10min at 65°C with the medium-stringency wash solution and discard the wash solution.
  14. Monitor the dot blot with a Geiger counter. Continue the washes if needed using the high-stringency wash solution. Do not let the membrane dry out or the probe will permanently adhere to the membrane.
  15. To quantitate each spot on the dot blot, expose the filter to a Phosphoimager cassette. Alternatively, employ X-ray film to identify labeled regions on the nylon membrane, excise each sample, and quantitate using a scintillation counter.
  16. Plot a standard curve of DNA concentration vs integrated intensity per counts per minute for the DNA standards and employ the curve to determine the concentration of DNA in fractions obtained from the heparin sulfate column. (Note: The replication center assay is also a useful method to calculate the rAAV titer. The rAAV particle number of each fraction can be calculated. Remember to take into consideration that plasmid standards are double stranded whereas rAAV virions harbor only a single strand.)

  1. HindIII is used in the digest to cut the rep and cap fragment in half for easy isolation of the plasmid backbone.
  2. DNA preparation should be pure. Purify your viral fragment by agarose gel separation and running onto Whatman DEAE-8 1 paper or a preparation of equivalent high quality.
  3. Alternatively, blunt-end ligation may be used to construct the rAAV vector plasmid.
  4. For efficient packaging into AAV capsids, the size of the rAAV construct (including the 190-bp ITRs) must be 4.6 kb or less.
  5. Plasmids are grown in the Sure strain of Escherichia coli. The literature shows that AAV ITRs are unstable in bacteria. To avoid deletion, restrict bacterial growth in the stationary phase. If you still obtain deletions, grow the plasmids at 30°C for only 12 h. The integrity of the plasmids can be assayed by restriction enzyme digests.
  6. Polypropylene and glass attract ionic strengthdependent aggregates more than polystyrene. For this reason, mixing containers made of polystyrene are preferred for transfections.
  7. The total DNA is equal to 37.5µg/plate, and the ratio of rAAV construct to the pXX2 and pXX6 is equal to a molecular ratio of about 1:1:1.
  8. If a coarse precipitate forms, decrease the incubation time. If a precipitate forms too quickly (i.e., less than I min) check the pH of the 2X HeBS. The pH range of 2X HeBs should be between 7.05 and 7.12.
  9. Only use sterile ddH2O. Do not autoclave!
  10. After 24 h, the 293 cells (when viewed through a microscope) should have a rounded appearance, indicating viral replication. If detached cells are noted, the incubation was too long.
  11. Cell suspensions or cell precipitates may be stored at -20°C for up to 6 months.
  12. The NaCl in the 15% iodixanol layer will separate viral aggregates that may form due to the high concentration of virus.
  13. Collecting fractions from the flow-through and washing steps ensures that all the virus was bound to the column and eluted as the salt gradient increased.
  14. Using a new heparin column for each purification ensures that you will not cross-contaminate viral preparations.

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