TITLE: AAV Purification Protocol with Iodixanol Gradient
AUTHORS: Roberta Marongiu

[DESCRIPTION]
Protocol used in the Kaplitt and Marongiu labs to purify AAVs.

[STEPS]
SECTION: Transfection
1. Plate 293 cells in CellSTACK double chamber in 200 mLmedia (DMEM with 10% FBS and 1% Penicillin/Streptomycin) so that at the moment of transfection they reach 80% confluence.

SECTION: Transfection
2. When the cells reach 80% confluence, they are ready to be transfected. While preparing the transfection reagents, change the incubator CO2 level to 3%.
SECTION: Transfection
3. Prepare transfection reagents and set aside at Room temperature: 2x Hepes Buffer Saline (HBS), sterile H2O and 2.5 M CaCl2 in water.

SECTION: Transfection
4. 
Prepare DNA/CaCl2 solution: when the cells are approximately 80% confluent, prepare total of 200-400 µg of DNA (1:1:1 molar ratio for pAAV and helper plasmids) in 10 mL CaCl2 solution in sterile H2O (CaCl2 final concentration 0.25M).

SECTION: Transfection
5. Prepare the transfection mix: Place 110 mL of 2x HBS in a 50mL tube.  Using a 5 mL pipette, bubble solution from the bottom of the tube while slowly adding the CaCl2/DNA mix.  Bubbles should appear at approximately the same rate at which the drops are released into the solution.  The final volume at the end of this step will be 20 mL 1/10 of the volume of the cells plated on the cell stacks.  If higher volumes of reagents are needed, the final volume can be adjusted according to this ratio.

SECTION: Transfection
6. Place a small droplet of the transfection mix on a small plate or slide and view under the microscope.  The DNA precipitates should look like small particles and be visible in the center of the drop.  If they look like large particles or aggregates, discard the mix and attempt to re-bubble the solution.  If unsuccessful, prepare new solutions and repeat.
SECTION: Transfection
7. Add the transfection mix to 200 mL of fresh 5% DMEM (DMEM with 5% FBS and 1% penicillin/streptomycin).

SECTION: Transfection
8. Gently remove media from the cell stack and replace it with the 220 mL 5% fresh media/transfection mix.  Swirl the stack gently and return it to the incubator.  Incubate for 1200 min to  1440 min; do not disturb the cells during this time.

SECTION: Transfection
9. Check the cells: the precipitate from the transfection mix should be visible on the cells and mostly in the areas of the stack with no cells attached.  Discard the media and add 50 mL of PBS or DMEM.  Gently rock the stack to wash and discard the solution.  Replace with 200 mL of fresh DMEM with 5% FBS and incubate at 5% CO2 for 2880 min.

SECTION: Harvesting
47. Resuspend pellet with 10 ml 1x GB buffer.
SECTION: Harvesting
10. Pour the media off the cell stack into a 500 mL centrifuge bottle. Separate three 50 mL aliquots of this media in conical tubes. Add 0.5 M EDTA to each of the first two tubes, to a final concentration of 10 mM (1-2 ml).

SECTION: Harvesting
10.1. i. Add the contents of one tube of media/EDTA to the cell stack and rock gently, tapping the sides of the stack to detach the cells. Return this media/EDTA back to the 50 mL conical tube.
SECTION: Harvesting
10.2. ii. Add the contents of the second tube of media/EDTA to the cell stack and rock gently until the cells come off the stack. It should take no longer than5 min. Return this media/EDTA back to the 50 mL conical tube.

SECTION: Harvesting
10.3. iii. Wash with the third tube of media, without EDTA. If the stack has cells wash with fresh media, HBSS or PBS.
SECTION: Harvesting
10.4. iv. Use the third tube to wash the first two (EDTA) tubes – cells cling to the tubes.
SECTION: Harvesting
10.5. v. Return the contents of all the conical tubes to the 500 mL centrifuge bottle and incubate for about 10 min in an on ice bucket with water/ice.

SECTION: Harvesting
10.6. vi. Spin down at 4 °C at 500x g for 15 min or 1000 x g for 10 min. If still floating cells are present, spin extra 10 min.

SECTION: Harvesting
10.7. vii. Pour off the media completely and, if necessary, use a pipette to remove any remaining media drops.It is very important that all media be removed.
SECTION: Harvesting
11. Resuspend pellet with 10 mL 1x GB buffer.

SECTION: Harvesting
12. Freeze -80 °C for at least 60 min if purifying on the same day (or until purification day). 

SECTION: Harvesting
13. Thaw in 42 °C° water bath.

SECTION: Harvesting
14. Sonicate: Wash sonication probe with H20, then 70% alcohol, then H20 again and wipe with a clean kimwipe. Lyse with a sonication probe: output control – 3, duty cycle % - 30.Sonicate for 6 to 10 beats. The probe should be ~1 cm from the tube bottom.
SECTION: Harvesting
15. Freeze in -80 °C for 60 min. Thaw in 42 °C water bath – Freeze/thaw like this 3x. (Can do last freeze O/N, and proceed to benzonase step next day).

SECTION: Harvesting
16. Bring sample to37 °C, add CaCl2 (use 4uL of 2.5M stock CaCl2) and then treat with Benzonase (2uL in 10mL) at 37 °C for 60 min-swirl every 15 minutes.

SECTION: Harvesting
16.1. CaCl2 (2.5M for transfection) – 2500x, so use 4Ll in 10mL.
SECTION: Harvesting
16.2. Benzonase (25 KU stock), need 500 units, so add 2uL to 10 mL.
SECTION: Harvesting
17. Centrifuge at 3000 x g for 15 min at 4 °C.

SECTION: Harvesting
18. Transfer supernatant to new tube, store at 4 °C 15 min (or continue on).

SECTION: Harvesting
19. Prepare optiprep gradients – Mix, store at 4 °C.

SECTION: Harvesting
20. Prepare gradients: 12-13 mL AAV, 6 mL 15%, 8 mL 25%, 8 mL 40%, 5 mL 58%

 
 
 
     To make >100 ml      15% + 1M NaCl      25%      40%      58%        Optiprep (60%)       40 ml      46.7      64      96.67        10x GB      16 ml      11.2      9.6      3.33        5M NaCl      32 ml      -      -      -        ddH2O      72 ml      54      22.4      -        Phenol Red (5mg/ul)      -      280 ul      -      240ul        TOTAL VOL:      160 ml      112 ml      96 ml      100 ml   

     To make ~25 ml      15% + 1M NaCl      25%      40%      58%        Optiprep (60%)       5 ml      11.68      16      24.17        10x GB      2 ml      2.8      2.4      .83        5M NaCl      4 ml      -      -      -        ddH2O      9 ml      13.5      5.6      -        Phenol Red (5mg/ul)      -      70 ul      -      60 ul        TOTAL VOL:      20 ml      28 ml      24 ml      25 ml   
 
 
 
SECTION: Harvesting
21. Centrifuge in ti70 rotor at 360,000g (70,000rpm) for 70 min, 18 °C, use acceleration and deceleration protocol #9.

SECTION: Harvesting
22. Collect virus fraction:
SECTION: Harvesting
22.1. i. Puncture tube at 58/40 interface with 18G needle attached to 10ml syringe.
SECTION: Harvesting
22.2. ii. Collect about 2 mL with bevel up and 2 ml with bevel down. AVOID PROTEIN BAND AT 40/25 INTERFACE.

SECTION: Harvesting
23. 
Equilibrate the Millipore® membrane in a 15 mL conical tube with 4 mL PBS-Mg: Add solution to the membrane and centrifuge at 2000 x rpm in TC room until all liquid has passed through (about 2 min).Check after 2 min if the liquid is going through at same speed in different columns. If there is one there is washing faster discard and take a new one (that means the column could be damaged). 

SECTION: Harvesting
24. Discard the PBS-Mg from bottom of the conical tube and load the virus solution. Centrifuge at the same speed until only 1 mL remains above the filter (about 10 min).Add 3 mL PBS-Mg and pipette up and down with a P1000 pipette without touching the membrane. Spin down as before until 1 mL remains. Repeat five times. During the last wash, spin down and leave only 300-500 μL above the membrane in which to recover the virus.

SECTION: Harvesting
25. Wash down the sides of the membrane by pipetting this volume. Transfer to a 1.5 mL Eppendorf tube. Wash twice with 100-200 μl PBS-Mg while scratching back and forth over the membrane with the pipette tip.
SECTION: Harvesting
26. Do a quick spin on the bench top centrifuge before sonication to have all the pieces of membrane on the bottom. Sonicate 6 bursts/beats (output control 1.5-2, duty cycle % - 30) to break up viral aggregates. Centrifuge at 5000 x g for 10 min at4 °C. Transfer supernatant to a new tube.

SECTION: Harvesting
27. Equilibrate a 0.22μm filter with 500 μl PBS-Mg. Aspirate the volume with a 18 1/2 GA needle
SECTION: Harvesting
28. Using the same needle and syringe, aspirate the virus.
SECTION: Harvesting
29. Gently pass the virus through the filter. Collect into a 1.5 mL Eppendorf tube.
SECTION: Tittering by RT-qPCR via standard curve method
31. Inactivate DNAse: Add 2 μl EDTA (25 mM stock solution) to the tube and incubate at 70 °C for 10 min.

SECTION: Tittering by RT-qPCR via standard curve method
32. Digest capsid: Add 10 μl 2 M NaOH and incubate at 56 °C for 20 min.  Then dilute immediately 1:100 in 10 mM Tris pH 8.0.

SECTION: Tittering by RT-qPCR via standard curve method
33. Standards: prepare standards as described in dedicated protocol.
Preparation of qPCR standards for AAV tittering  .

SECTION: Tittering by RT-qPCR via standard curve method
30. Prepare DNA probe: 2 μl virus, 1 μl 10x DNAse 1 buffer with MgCl2, 1 μl DNAse 1, and 6 μl ddH2O in a PCR tube. Incubate at 37 °C for 15 minto   60 min.