Jul 23, 2020
AAV production for Serotypes with Heparin Binding Capabilities
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Sierra Simpson, Olivier George 2020. AAV production for Serotypes with Heparin Binding Capabilities. protocols.io https://protocols.io/view/aav-production-for-serotypes-with-heparin-binding-82zhyf6
Manuscript citation:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3308604, /https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418430/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 05, 2019
Last Modified: July 23, 2020
Protocol Integer ID: 29497
Abstract
Production of Adeno Associated Virus with Heparin Binding Motifs. Adapted from McClure et. al.
Materials
MATERIALS
Tris
Sodium ChlorideCatalog #PubChem CID: 5234
Sodium DeoxycholateCatalog #PubChem CID: 23668196
100 ml PolyethyleniminebiorbytCatalog #orb65580
HiTrap Heparin HP affinity columnGe Life SciencesCatalog #17040701
Benzonase® NucleaseSigma-aldrichCatalog #E1014 SIGMA
DMEMInvitrogen - Thermo Fisher
Amicon Ultra Centrifugal Filter (100K)Emd MilliporeCatalog #UFC810096
Cell scraperCorningCatalog #3011
Plasmids Needed Per Prep:
62.5 ug AAV plasmid
125 ug pHelper
62.5 ug pAAV DJ
Safety warnings
AAV is BSL1, but should be handled with caution and kept in a hood when processing.
Before start
Plate HEK293T cells to 70-80% confluency in 5 * 15 cm Nunc tissue culture dishes for one batch of virus ( enough for injecting 10-20 animals. )
Transfection
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
In a separate tube mix 350ul PEI in 10.5ml MEM (PEI stock solution is 1mg/ml)
Wait 5 min then mix the two solution together
Incubate for 15 min at RT.
Volumize to 100ml with complete DMEM
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
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.
Wash cells with warm (37C) 1x PBS 10ml.
Add 20 mL warm PBS to each plate and gently remove cells with cell scraper.
Collect suspension in 50 mL falcon tubes.
Pellet cells at 800 x g for 10 min
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.
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.
Incubate at 37 deg C for 1 hour.
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.
Freeze supernatant at -20 deg C for 24 hours. ( This is important and should not be skipped - it allows for better isolation of virus )
Thaw solution, mix thoroughly and incubate at 37 deg C for 1 hour.
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
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).
Equilibrate column with 10 mL 150 mM NaCl, 20 mM Tris, pH 8.
Apply 50 mL virus solution to column and allow to flow through.
Wash column with 20 mL 100 mM NaCl, 20 mM Tris, pH 8.
Using 5 mL syringe continue to wash column with 2 mL 200 mM NaCl, 20 mM Tris, pH 8. Discard flow through.
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).
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
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.
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.
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).
Filter vector through 13 mm diameter 0.2 um syringe, aliquot and store at -80C.