TITLE: AAV production for Serotypes with Heparin Binding Capabilities
AUTHORS: Sierra Simpson, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Production of Adeno Associated Virus with Heparin Binding Motifs. Adapted from McClure et. al. </div></div>

[STEPS]
?. [Transfection]
Mix DNA in 4.5ml MEM as follow 
62.5 ug AAV plasmid 

125 ug pHelper 

62.5 ug pAAV DJ 

Filter combined plasmids using 0.22 um filter
?. [Transfection]
In a separate tube mix 350ul PEI in 10.5ml MEM (PEI stock solution is 1mg/ml)
?. [Transfection]
Wait 5 min then mix the two solution together
?. [Transfection]
Incubate for 15 min at RT.
?. [Transfection]
Volumize to 100ml with complete DMEM
?. [Transfection]
One by one, remove culture medium from each dishes and gently add the fresh transfection solution (20ml for each dishes).
?. [Lysing of cells and harvesting of AAVs]
48 to 72 hours after transfection (depending on health of the HEK cells), remove media from cell culture plates and discard. The cells will still exhibit nice monolayer formation - of cells start to peel away from the bottom of the flask it is too late. Watch for media turning from pink to yellow. If media starts to look yellow, it is time to wash.
?. [Lysing of cells and harvesting of AAVs]
Wash cells with warm (37C) 1x PBS 10ml.
?. [Lysing of cells and harvesting of AAVs]
Add 20 mL warm PBS to each plate and gently remove cells with cell scraper.
?. [Lysing of cells and harvesting of AAVs]
Collect suspension in 50 mL falcon tubes.
?. [Lysing of cells and harvesting of AAVs]
Pellet cells at 800 x g for 10 min
?. [Lysing of cells and harvesting of AAVs]
Discard supernatant and resuspend pellet in 150 mM NaCl, 20 mM Tris pH 8.0 using 10 mL per culture plate. Split into two 50 mL tubes.
?. [Lysing of cells and harvesting of AAVs]
Prepare fresh solution of 10% sodium deoxycholate in ddH2O. Add 1.25 mL of this to each tube for final concentration of 0.5%. Add benzonase nuclease for final concentration of 50 units/mL. Mix thoroughly.
?. [Lysing of cells and harvesting of AAVs]
Incubate at 37 deg C for 1 hour.
?. [Lysing of cells and harvesting of AAVs]
Remove cellular debris by centrifuging at 3000 x g for 15 min. Transfer supernatant to fresh 50 mL tube and ensure all cell debris has been removed.
?. [Lysing of cells and harvesting of AAVs]
Freeze supernatant at -20 deg C for 24 hours. ( This is important and should not be skipped - it allows for better isolation of virus )
?. [Lysing of cells and harvesting of AAVs]
Thaw solution, mix thoroughly and incubate at 37 deg C for 1 hour.
?. [Lysing of cells and harvesting of AAVs]
Centrifuge at 3000 x g for 15 min. Transfer supernatant to fresh 50 mL tube and ensure all cell debris has been removed to avoid blocking heparin columns.
?. [Heparin Column Purification ]
Setup HiTrap heparin columns with peristaltic pump so solutions flow through at 1 mL/minute. Avoid air bubbles in heparin column (and tubes).
?. [Heparin Column Purification ]
Equilibrate column with 10 mL 150 mM NaCl, 20 mM Tris, pH 8.
?. [Heparin Column Purification ]
Apply 50 mL virus solution to column and allow to flow through.
?. [Heparin Column Purification ]
Wash column with 20 mL 100 mM NaCl, 20 mM Tris, pH 8.
?. [Heparin Column Purification ]
Using 5 mL syringe continue to wash column with 2 mL 200 mM NaCl, 20 mM Tris, pH 8. Discard flow through.
?. [Heparin Column Purification ]
Elute virus into 15 mL centrifuge tube with following steps (Binding affinity of DJ capsid slightly differ from the one of AAV2, see Thomas F. Lerch et al., Strucure 2012).
?. [Heparin Column Purification ]
Using 5 mL syringe and gentle pressure, apply  the following in order: 1. 1.5mL 300mM NaCl, 20mMTris, pH 8 2. 3mL 350mM NaCl, 20mM Tris, pH 8 3. 1.5mL 450mM NaCl, 20mM Tris , pH8
?. [Concentration and sterile filtration of rAAVs]
Concentrate vector using Amicon ultra-4 centrifuge with 100000 molecular weight cutoff. Load 4 mL of column eluate into concentrator and centrifuge at 2000 x g for 2 min at RT. Discard flowthrough and reload concentrator with remaining virus solution and repeat centrifugation.
?. [Concentration and sterile filtration of rAAVs]
Concentrated volume should be 250 uL. If concentrated volume is significantly more than this, discard the flow through and continue to centrifuge in 1 min steps until volume is approximately 250 uL.
?. [Concentration and sterile filtration of rAAVs]
Add 250 uL of PBS to virus for a final volume of 500 uL and remove from concentrator (this step could be skipped if high titer is required).
?. [Concentration and sterile filtration of rAAVs]
Filter vector through 13 mm diameter 0.2 um syringe, aliquot and store at -80C.