Download Protcol for Adeno- Associated Virus (AAV) production

Protocol for Adeno- Associated Virus (AAV) production
Advantages of gene delivery through the use of Adeno-Associated Virus (AAV) are
nonimmunogenicity to host, nonpathogenic property, broad cell and tissue tropism and a
possibility to integrate with genome only in specific sites. Recombinant AAV virus is
constructed by replacing cap and rep genes coding capside proteins and integration to
genome assistance proteins by transgene. Rep and cap genes need to be provided on a
helper plasmid pDP. There are 5 serotype of pDP which provides different capside
proteins, what leads to different tissue and cells tropism. Helper1 pDP1 has primary
tissue tropism to: human spine, eye, brain, liver, nose, muscle and mouse, brain; Helper2
pDP2 to: human eye, spine, liver, nose, and monkey kidney and Helper5 pDP5 to: human
heart, nose, muscle and rat liver.
Materials per 2 T-175 flask
Plasmids




AAV-Syn-EGFP (or other plasmid of interest, which should be approx in
equimolar ratio wit Helper1/ Helper2 mix): 20 ug
Helper1 pDP1: 35 ug
Helper2 pDP2: 35 ug
Or use Helper5 pDP5 instead of pDP1-pDP2 mix
Materials
 PEI (1ug/ul) – Polyethylenimine 25kD linear from Polysciences (cat. 23966-2)
Preparing stock solution:
Dissolve PEI in endotoxin-free dH2O (needs to be heated to 80°C)
Let cool to room temperature
Neutralize to pH 7.0, filter sterilize (0.22 um), aliquot and store in -20°C, working stock
in 4°C.
 DMEM medium without additives (for transfection)
 DMEM medium with
(for cell growth and virus production)
L-glutamine
antibiotics: penicillin, streptavidin
10%FBS
 T-175 flask (2 per single virus)
 Whatman filters FP 30/0.45 um
 Amicon Ultra-15 protein concentration columns 50K Millipore cat. UFC905024
 Cell Lysis Buffer
50 mM TrisCl pH 8.0
150 mM NaCl
filtered sterile
 Benzonase Nuclease (Sigma, cat. E1014)
 Benzonase Buffer 10x
500 mM TrisCl pH 8.0,

10 mM MgCl2,
1mg/ml BSA filtered sterile
Phosphate Buffer Saline PBS without Ca and Mg
HEK 293FT cell preparation
 Prepare HEK 293FT cell at the confluence of approx 70-80% (5 million of cells
per T175 flask should be plated a day before the transfection)
Transfection of HEK 293FT cells
1. DNA and PEI mix preparation
 Mix AAV-Syn-EGFP/pDP1/pDp2 in 1.5 ml of DMEM (no serum)
 Mix 270 ul of PEI in 1.5 ml of DMEM (no serum)
Add PEI mix to DNA mix (PEI/DNA should be 3:1 ratio) incubate at least 15 min. at
RT
2. Add DMEM (no serum) to PEI/DNA mix up to 15 ml
3. Replace the medium on HEK 293 FT cells with PEI/DNA/DMEM mix (7.5 ml per
flask)
4. Incubate 4-5 h at 37°C, 5% CO2
5. Change medium to DMEM with additives
6. Incubate 48-72 h at 37°C, 5% CO2
Harvesting of the virus
1. Gently wash the cells with PBS
2. Add 10-15 ml of PBS and incubate for 5 min at 37°C, 5% CO2 (Since calcium is
not present in PBS cells loose the adhesion and begin to drop from the plastic)
3. Collect cells in 50 ml Falcon tube and spin at 1000 rpm for 5 min
4. Remove supernatant.
5. Dissolve cells in 2 ml of Cell Lysis Buffer
6. Freeze/thaw 3x with the shaking of cells every time.
7. After last thawing spin down the cell pellet at 2000 rpm for 5 min
8. Benzonase treatment: add respective amound of 10x Benzonase Buffer to make
1x and about 250 units of Benzonase per one prep, incubate 30 min. at 37°C, 5%
CO2
9. Spin down again at 2000-2500 rpm for 5 min.
10. Take the supernatant and filter through 0.45 uM filter using syringe
11. Increase the volume of the viral prep up to 15 ml wit 150 mM NaCl
12. Load the viral prep on the Amicon Ultra column
13. Spin down 4000 rpm, 1-2 h at +4°C until the 200 ul volume is reached
14. Aliqout approx 10-15 ul per sample, store at -80°C, working aliquot at +4°C up to
1 month
References:
YANG LU, STEM CELLS AND DEVELOPMENT 13:133–145 (2004), “Recombinant
Adeno-Associated Virus As Delivery Vector for Gene Therapy—A Review”