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.
II. MATERIALS AND INSTRUMENTATION
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)
Plasmid with transgene of interest
Plasmid pXX2, the AAV helper plasmid (Samulski laboratory); map and sequence are available on the internet at http://www.med.unc.edu/genether/
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
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
2. Transfection of 293 cells
Note: Warm up 2X HeBS buffer, 2.5 M CaCl, and filtered distilled water to room temperature.
3. Cell Harvesting
Note: Harvesting should occur within 64 h but not less than 48 h of transfection.
4. Purification of Recombinant Virus
5. Purification Using an Iodixanol (Optiprep) Gradient
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.
7. Delivery of Recombinant Virus in Vitro
a. Determination of rAAV titer by Dot Blot Assay
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