TITLE: AAV Production and Purification
AUTHORS: Justin T Savage

[DESCRIPTION]
This protocol is for the production and purification of Adeno-Associated Virus. The protocol contains the necessary steps to produce AAVs from HEK293T cell cultures. 

[STEPS]
SECTION: Growing HEK 293T Cells: Day 1 -Morning
1. Make Media for HEK293T Cells
SECTION: Growing HEK 293T Cells: Day 1 -Morning
2. Thaw HEK293T Cells in 10cm Dish with 10 ml HEK Medium (One Vial into three 10cm Dish)
SECTION: Day 3 -Morning
3. Split HEK293T Cells (Usually become confluent on Day 3) with 3ml Trypsin.
SECTION: Day 3 -Morning
4. Use serum to block Trypsin (100ul for 1ml trypsin)
SECTION: Day 3 -Morning
5. Split one full confluent 10cm dish into five 10cm plates (So Total 15 Plates).
SECTION: Day 4 - Evening
6. Split all plates of HEK293T Cells (15 10 cm dish confluent plates)
SECTION: Day 4 - Evening
7. Plate 15 million cells/T175 Flask with 20 ml Medium
SECTION: Growing HEK 293T Cells: Day 1 -Morning
1.1. 500ml DMEM
5ml 100x Pyruvate
5ml 100x Pen/Strep
5ml 100x L-Glutamine
50ml Fetal Bovine Serum
SECTION: Transfection: Day 5 - Evening
8. Make sure all plasmids are ready on day of Transfection
SECTION: Transfection: Day 5 - Evening
9. In a sterile tube, dilute total plasmid DNA (ug) in 6ml Opti-MEM.
SECTION: Transfection: Day 5 - Evening
10. In another sterile tube, dilute 1.33ml PEI (7.5uM) in 4.66ml Opti-MEM.
SECTION: Transfection: Day 5 - Evening
11. Incubate 10 minutes at RT.
SECTION: Transfection: Day 5 - Evening
12. Combine the two tubes, mix well and incubate 20 minutes at RT.
SECTION: Transfection: Day 5 - Evening
13. Add 2ml of DNA/PEI mixture to each plate of cells, mix well and return to incubator.
SECTION: Day 6 - Morning
14. Change media (Usually 6-18 hours later)
SECTION: Day 6 - Morning
15. Add 20ml fresh media and culture for 72hrs
SECTION: Transfection: Day 5 - Evening
8.1. pAd-DeltaF (Order from AddGene; Glycerol Stock-Cultivate at 30°C and do Maxi) 
PHP.eB Capsid (Order from AddGene; Glycerol Stock-Cultivate at 37°C and do Maxi)
ITR-Gene of interest plasmid (i.e. GEARBOCS with gRNA for gene of interest)
SECTION: Transfection: Day 5 - Evening
9.1. 30 ug pAd-DeltaF
15 ug PHP.eB
15 ug ITR-Gene of interest (pAAV2ITR-gfaABC1D-XXX)
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
16. Collect the cells and medium by scraping the cells off the dish with a cell scraper and transferring it to a 50-ml conical tube.
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
17. Rinse dishes with 2 ml of 1× PBS and transfer it to the same conical tube.
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
18. Harvest two dishes at a time into the same 50-ml tube
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
19. Centrifuge at 1300 rpm for 8 min in a tabletop centrifuge.
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
20. Aspirate the medium from the conical tube and repeat cell collection steps until cells are pelleted from all 6 dishes into one tube. The same tube can be used for pelleting cells from all the dishes.
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
21. Resuspend the final pellet from all 6 dishes in 4 ml of cell lysis buffer (In TC Room Fridge-50 ml Tube) (4 ml for one virus).
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
22. Prepare dry ice/ethanol bath during the last spin
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
23. Take dry ice in ice box and add 100% 190 proof ethanol to that to make dry ice/ethanol bath.
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
24. Freeze the pellet in the dry ice/ethanol bath and thaw in a 37 °C water bath three times (Almost 10-minute incubation each).
SECTION: Collecting Cell Lysate: Day 8 - Morning or Noon
25. Freeze again in dry ice/ethanol bath and store at -80 °C. The pellet can be stored  at −80 °C indefinitely and thawed at your convenience.
SECTION: Purifying Virus: Day of convenience - Morning
26. Hydrolyze nucleic acids
SECTION: Purifying Virus: Day of convenience - Morning
26.1. Thaw cell lysate in a 37 °C water bath
Add 8ul of Benzonase to the 4 ml of thawed cell lysate at a final concentration of 50 U/ml (Benzonase stock conc. 25U/ul).
Incubate at 37 °C in a water bath for 30 min.
Centrifuge lysed cells at 4500 rcf for 30 min at 4 °C in a tabletop centrifuge
SECTION: Purifying Virus: Day of convenience - Morning
27. Prepare the iodixanol gradient during the Centrifugation.
SECTION: Purifying Virus: Day of convenience - Morning
27.1. The following volumes are for two gradients (or two viruses) using OptiSeal tubes. (Note: Always keep one extra. i.e if you have three viruses make gradient solutions for four)
SECTION: Purifying Virus: Day of convenience - Morning
27.2. Take four 50ml Falcon Tubes and label well each concentration of iodixanol
SECTION: Purifying Virus: Day of convenience - Morning
27.3. Add each component carefully to each tube and mix well by vortexing: 

Iodixanol Gradient for two Viruses (Make one extra if you have 3 or more viruses)
 

SECTION: Purifying Virus: Day of convenience - Morning
27.4. Use a 10 or 12 ml syringe with an 18 G needle to add these solutions to each labelled centrifuge Optiseal tube in the order below.
(Notes before you add: Add very slowly along the sides of the tube and take care to avoid bubbles. The same needle can be used for loading all steps. Take exact volume otherwise 4ml virus cannot be accommodated over the top in last step)

i. 5 ml of 60% iodixanol (Bottom)
ii. 5 ml of 40% iodixanol
iii. 6 ml of 25% iodixanol
iv. 9 ml of 15% iodixanol step (Top)
SECTION: Purifying Virus: Day of convenience - Morning
27.5. Collect vector-containing supernatant from centrifuged tube using 10 or 12- ml syringe with 18G needle. The volume of the supernatant is approximately 4-4.5 ml.
SECTION: Purifying Virus: Day of convenience - Morning
27.6. Load the vector-containing supernatant over the iodixanol density gradient prepared before
SECTION: Purifying Virus: Day of convenience - Morning
27.7. Fill to the very top until the hinge of the tube. If you have space left over, top off the tube with cell lysis buffer but only until the hinge.
SECTION: Purifying Virus: Day of convenience - Morning
27.8. Close with the dark plug provided along with the Optiseal tube without anair bubble. If your tube does collapse and is hard to remove from the rotor, then drip acetone between the tube and rotor wall and extract with tweezers
SECTION: Purifying Virus: Day of convenience - Morning
28. Ultracentrifugation (67,000 rpm for 1 hour at 18°C)
SECTION: Purifying Virus: Day of convenience - Morning
28.1. Bring Beckman Ti70 rotor
Put aerocap over the Optiseal Tubes
Keep tubes inside the rotor and press down
Tighten the lid and bring to the Beckman ultracentrifuge and keep inside
Close the lid and press vaccum (Green light blinks)
Press Speed-Press 67000-Press Enter
Press Temp- Press 18°C-Press Enter
Press Time- Press 1.00hr- Press Enter
After the vacuum green light is constant without blinking-Press Enter-Press START
Wait until it reaches the maximum speed before leaving centrifuge
SECTION: Virus Collection: Same Day as Virus Purification
29. Set up ring stand and clamp inside the hood to hold tube
SECTION: Virus Collection: Same Day as Virus Purification
30. Prepare bleach in bottle and keep inside the hood to discard tips and tubes
SECTION: Virus Collection: Same Day as Virus Purification
31. Once the centrifugation is done, bring rotor to TC room and open
SECTION: Virus Collection: Same Day as Virus Purification
32. Remove the aerocap from each tube using a pair of pair pliers
SECTION: Virus Collection: Same Day as Virus Purification
33. Remove black plug from each tube and discard into bleach bottle
SECTION: Virus Collection: Same Day as Virus Purification
34. Take out the Optiseal Tube with pliers by clipping the topside and place inside the hood
SECTION: Virus Collection: Same Day as Virus Purification
35. Spray down 70% alcohol to player, aerocap and inside rotor; and keep for 10 minute and wipe with tissue paper.
SECTION: Virus Collection: Same Day as Virus Purification
36. Keep ready the Millipore Amicon filter unit (UFC910008, 100K MWCO) with proper label
SECTION: Virus Collection: Same Day as Virus Purification
37. Keep the optiseal tube in the holder and tight well.
SECTION: Virus Collection: Same Day as Virus Purification
38. Lay a bunch of tissue paper down and keep the bleach bottle.
SECTION: Virus Collection: Same Day as Virus Purification
39. Puncture the tube on the side slightly below (3–5 mm) the 60–40% interface with an 18-gauge needle (bevel up) attached to a 10 ml syringe.
SECTION: Virus Collection: Same Day as Virus Purification
40. Slowly draw the 2-3 ml viral solution out from clear zone from each centrifuge tube by aspiration using the needle and put into the filter unit.
SECTION: Virus Collection: Same Day as Virus Purification
41. CRITICAL STEP: Avoid the proteinaceous material near the 40–25% interface.
SECTION: Virus Concentration: Same Day as Virus Purification
42. Add 1ml of DPBS (#14190-144-Ca/MgCl2 Free) to filter unit and mix well using P1000
SECTION: Virus Concentration: Same Day as Virus Purification
43. Spin 4000g for 10 minutes.
SECTION: Virus Concentration: Same Day as Virus Purification
44. Repeat steps 43 and 44 five times.
SECTION: Virus Concentration: Same Day as Virus Purification
45. In last step, concentrate up to 150-200ul
SECTION: Virus Concentration: Same Day as Virus Purification
46. Collect virus from filter unit using P200 into a 1.6 ml Eppendorf tube
SECTION: Virus Concentration: Same Day as Virus Purification
47. Aliquot the virus in 10ul amount to tubes and freeze in liquid N2
SECTION: Virus Concentration: Same Day as Virus Purification
48. Store at –80 °C indefinitely