Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification
- HEK293 Cells (ATCC CLR-1573)
- Dual or Tri-Plasmid AAV System (Chamberlain )
- Dulbecco’s Modified Essential Media (Life Technologies 11995-073)
- Fetal Bovine Serum (HyClone SH30071.03)
- L-Glutamine (100 ×) (Life Technologies 25030-081)
- Antibiotic-Antimycotic (100 ×) (Life Technologies 15240112)
- MEM Non-Essential Amino Acids (100 ×) (Life Technologies 11140-050)
- 200 mM Magnesium Chloride (Sigma M8266)
- Giga-Scale Plasmid Purification System (Qiagen 12991)
- PBS (1 ×) (HyClone SH30256)
- Benzonase Endonuclease (Sigma E1014-25KU)
- Protease Inhibitor (100 ×) (Sigma P2714)
- RNAse A (Sigma R4875)
- Tris Acetate-EDTA (10 ×) (Sigma T8280)
- Calcium Chloride Hexahydrate (Sigma 442909)
- HyClone Water, Molecular Biology Grade (Thermo Scientific SH30538)
- N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic Acid (BES) (Sigma B4554)
- Sodium Phosphate Dibasic (Sigma S3264)
- Sodium Chloride (Sigma S5886)
- Polyethylene Glycol 8000 (PEG8000) (Fisher Scientific BP233-1)
Cell culture media
Calcium phosphate precipitation reagents
10 × citric saline cellular detachment solution
- 1.35 M KCl
- 0.15 M sodium citrate
Precipitation reagent stock solution (PEG/NaCl) (5 ×)
Pellet suspension buffer
- Forma Steri-Cult CO2 Incubators (Thermo Scientific 3311) (Humidified, 5.0% CO2 regulated)
- Microfluidizer (Microfluidics M-110L Pneumatic)(Chilled on wet ice, 200micron Lysis Chamber, 9000psi Chamber Pressure)
- Li-Cor Odyssey Imager (Li-Cor)
- High Performance Centrifuge (Beckman Coulter J Series)
- JA-14 Rotor (Beckman #339247)
- Ultra-Centrifuge (Beckman Coulter Optima Series)
- SW-32 Rotor (Beckman Coulter)
- Ultra-Clear Thin-Walled Centrifuge Tube (Beckman Coulter 344058)
- Class II Bio-Safety Cabinet (Thermo Scientific EN12469)
- Temperature Controlled Water Bath (Fisher Isotemp 15-462-10Q)
- Tissue Culture Table Top Centrifuge (Sorval Legend RT 75004377)
- Swinging Bucket Rotor (Sorval 75006445)
- 500 ml Bucket Adaptor (Heraeus 75006441L)
- Bottle Rotor Cushions/Adaptors (Corning 431124)
- 10 Tray Cell Factory (Nunc EasyFill 140400)
- 1 Tray Cell Factory (Nunc EasyFill 140000)
- 150 ml 0.22 µm Express Plus Steritop Filter Unit (Millipore SCGPT01RE)
- 1000 ml Stericup Receiver Flask (Millipore SC00B10RE)
- 50 ml Conical Tube (Corning 430291)
- 250 ml Conical Tube (Corning 430776)
- 500 ml Conical Tube (Corning 431123)
1.Cell Factory seeding and calcium phosphate transfection
1.1.Prepare 2.4 × 108 HEK293 cells in 1.0 L 37°C Cell culture seeding media. Pipette 90 ml cell suspension into the 1TCF.
1.2.Pour the remaining cell suspension into the 10TCF. Evenly distribute cell suspension across all layers.
1.3.Incubate cell factories overnight at 37°C/5.0% CO2.
1.4.Ensure Cell Factory monolayer is at optimal 70% confluence.
1.5.Combine Transfection Reagent A solutions to a final volume of 60 ml.
1.6.In a second tube, aliquot 60 ml of Transfection Reagent B.
1.7.Add Transfection Mix A to Transfection B.
1.8.Incubate CaPO4 mixture for five to thirty minutes at room temp. Precipitate formation should be monitored by visualizing 500 ml aliquots under 40 × magnification collected at 5–10 min intervals throughout the incubation. The mix should be applied to cells when it is visually very opaque and precipitate appears as fine granules under magnification.
1.9.Pipette 10 ml transfectant into the 1TCF. Immediately pour the remaining mix into the 10TCF and distribute evenly across layers. Return to incubator and incubate between 16 h and 20 h.
1.10.Gently remove cell factories from incubator and decant media/transfection mix. Ensure minimal cell loss occurs.
1.11.Replace transfectant media with 800 ml 37°C Cell Culture Serum Exchange Media in the 10TCF and 80 ml in the 1TCF. Evenly distribute media across layers.
1.12.Return cell factories to incubator for 48 h.
2.Cell and Media Harvest
2.1.Remove cell factories from incubator and pool both into a 1.0 L collection flask.
2.2.Detach remaining cells by applying 50 ml 2x citric saline detachment solution to 10TCF. Evenly distribute across trays and incubate with rocking at room temp for 5–10 min. Use 5.0 ml for the 1TCF.
2.3.Visualize by eye cells have detached and pour into pooled supernatant receiver flask. Rinse Cell Factory three times with 50 ml unsupplemented DMEM.
2.4.The viral supernatant may be processed immediately or stored at -80°C until purification.
3.Crude lysate processing
3.1.Remove viral supernatant from freezer and thaw in 3.0 L beaker to catch any flask leakage. Viral lysate placed at ~25°C will thaw completely in 20 h.
3.2.Chill a microfluidizer on wet ice previous to cell lysis. Run crude supernatant through microfluidizer according to manufactures instructions.
3.3.Collect viral lysate in new 1.0 L Stericup Receiver Flask. Rinse fluidizer with 50 ml DMEM.
3.4.Vacuum filter viral lysate through 150 ml 0.22 µm Express Plus Steritop Filter Unit into one 1000 ml Stericup Receiver Flask used for collection. The filter may need to be changed up to ten times.
3.5.Add 10 ml Protease Inhibitor Cocktail, 10 U/ml Benzonase and 10 mg/ml RNase A to the clarified viral lysate. Incubate for 3 h in 37°C water bath.
3.6.Perform a second filter clarification identical to step 3.4. This clarification step should only require a few filter flasks.
4.Precipitation and purification
4.1.Add 250 ml 1 × Precipitation Reagent Stock Solution to lysate containing flask and incubate on ice for 90 min.
4.2.Transfer crude lysate/PEG mixture into two 500 ml conical tubes. The tubes can accommodate a maximum volume of ~650 ml (Crude Lysate in Fig. 2).
4.3.Spin in bench top centrifuge at 3000 × g 4°C for 30 min.
4.4.Remove and discard aqueous layer ensuring minimal pellet disruption (Aqueous Layer A in Fig. 2).
4.5.Resuspend crude viral pellet in 40 ml Pellet Suspension Buffer and aliquot into 50 ml conical.
4.6.Rinse tubes twice with Pellet Suspension Buffer and combine with pellet suspension (Crude Suspension in Fig. 2).
4.7.Clarify pellet suspension by centrifugation at 10,000 × g and 4°C for 10 min.
4.8.Transfer the viral containing aqueous layer to a Beckmann Ultra Clear SW32 tube and spin at 149,000 × g/4°C for 3 h. Discard aqueous layer ensuring minimal pellet disruption (Aqueous Layer B in Fig. 2).
4.9.Resuspend pure viral pellet in 5.0–10.0 ml Pellet Suspension Buffer (Purified AAV in Fig. 2).
- Blankinship MJ, Gregorevic P, Allen JM, Harper SQ, Harper H, et al. (2004) Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6. Mol Ther 10: 671-678. doi: 10.1016/j.ymthe.2004.07.016. [View Article] [PubMed] [Google Scholar]
- Wang J, Faust SM, Rabinowitz JE (2010) The next step in gene delivery: molecular engineering of adeno-associated virus serotypes. J Mol Cell Cardiol 50: 793-802. doi: 10.1016/j.yjmcc.2010.10.017. [View Article] [PubMed] [Google Scholar]
- Bish LT, Morine K, Sleeper MM, Sanmiguel J, Wu D, et al. (2008) Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Hum Gene Ther 19: 1359-1368. doi: 10.1089/hum.2008.123. [View Article] [PubMed] [Google Scholar]
- Zincarelli C, Soltys S, Rengo G, Rabinowitz JE (2008) Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16: 1073-1080. doi: 10.1038/mt.2008.76. [View Article] [PubMed] [Google Scholar]
- Palomeque J, Chemaly ER, Colosi P, Wellman JA, Zhou S, et al. (2007) Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo. Gene Ther 14: 989-997. doi: 10.1038/sj.gt.3302895. [View Article] [PubMed] [Google Scholar]
- Asokan A, Samulski RJ (2013) An Emerging Adeno-Associated Viral Vector Pipeline for Cardiac Gene Therapy. Hum Gene Ther 24: 906-913. doi: 10.1089/hum.2013.2515. [View Article] [PubMed] [Google Scholar]
- Wang W, Barnabei MS, Asp ML, Heinis FI, Arden E, et al. (2013) Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. Nat Med 19: 305-312. doi: 10.1038/nm.3079. [View Article] [PubMed] [Google Scholar]
- Guo P, Xiao X, El-Gohary Y, Paredes J, Prasadan K, et al. (2012) A simplified purification method for AAV variant by polyethylene glycol aqueous two-phase partitioning. Bioengineered 4: 103-106. doi: 10.4161/bioe.22293. [View Article] [PubMed] [Google Scholar]
- Guo P, El-Gohary Y, Prasadan K, Shiota C, Xiao X, et al. (2012) Rapid and simplified purification of recombinant adeno-associated virus. J Virol Methods 183: 139-146. doi: 10.1016/j.jviromet.2012.04.004. [View Article] [PubMed] [Google Scholar]
- Hajitou A, Rangel R, Trepel M, Soghomonyan S, Gelovani JG, et al. (2007) Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat Protoc 2: 523-531. doi: 10.1038/nprot.2007.51. [View Article] [PubMed] [Google Scholar]
- Miyake K, Miyake N, Yamazaki Y, Shimada T, Hirai Y (2012) Serotype-independent Method of Recombinant Adeno-associated Virus (AAV) Vector Production and Purification. J Nippon Med Sch 79: 394-402. [PubMed] [Google Scholar]
- Yang L, Jiang J, Drouin LM, Agbandje-McKenna M, Chen C, et al. (2009) A myocardium tropic adeno-associated virus (AAV) evolved by DNA shuffling and in vivo selection. Proc Natl Acad Sci U S A 106: 3946-3951. doi: 10.1073/pnas.0813207106. [View Article] [PubMed] [Google Scholar]
- Grieger JC, Soltys SM, Samulski RJ (2015) Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol Ther 24: 287-297. doi: 10.1038/mt.2015.187. [View Article] [PubMed] [Google Scholar]