TITLE: AAV production and purification
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

*IMPORTANT*
This protocol is based on Challis et al., Nat Protoc 14: 379, published in 2019. Since that publication, we have made a number of modifications to the protocol to increase throughput or yield. With these modifications, we routinely achieve yields of >5e12 vg per 150 cm dish, with a well packaging capsid (e.g. AAV9, AAV-PHP.eB, and AAV.CAP-B10). 

In Challis et al., Nat Protoc 14: 379, a Type 70 Ti rotor is used for ultracentrifugation of the AAV through a density gradient (steps 32-36). This rotor can hold 8 tubes, allowing for simultaneous purification of 8 separate AAV variants, at 1-10 plates (150 mm plates) per variant. To increase throughput, we also provide a smaller scale purification workflow with a Type 70.1 Ti rotor, which can allow for purification of 12 separate AAV variants, at 1-3 plates (150 mm plates) per variant. 

Significant modifications to the referenced protocol are indicated by *MODIFICATION*. Read and understand the original protocol, before following a modified protocol. Following the original protocol should also yield expected results. 

[GUIDELINES]
This protocol is based on Challis et al., Nat Protoc 14: 379, published in 2019. Since that publication, we have made a number of modifications to the protocol to increase throughput or yield. With these modifications, we routinely achieve yields of >5e12 vg per 150 cm dish, with a well packaging capsid (e.g. AAV9, AAV-PHP.eB, and AAV.CAP-B10). 

In Challis et al., Nat Protoc 14: 379, a Type 70 Ti rotor is used for ultracentrifugation of the AAV through a density gradient (steps 32-36). This rotor can hold 8 tubes, allowing for simultaneous purification of 8 separate AAV variants, at 1-10 plates (150 mm plates) per variant. To increase throughput, we also provide a smaller scale purification workflow with a Type 70.1 Ti rotor, which can allow for purification of 12 separate AAV variants, at about 1-3 plates (150 mm plates) per variant. 

Significant modifications to the referenced protocol are indicated by *MODIFICATION*. Read and understand the original protocol, before following a modified protocol. Following the original protocol should also yield expected results. 

[STEPS]
SECTION: Reagent setup: Plasmid DNA
1. 
 

Grow bacterial stocks in LB or Plasmid+ media containing the appropriate selective antibiotic; refer
to the Addgene catalog for suggested growth conditions. Use a large-scale endotoxin-free plasmid purification kit to isolate plasmids; elute plasmid DNA with the supplied Tris-EDTA (TE) buffer. Measure the DNA purity and concentration using a spectrophotometer and freeze at -20 °C or
-80 °C for up to several years. 

 

SECTION: Reagent setup: Cell culture media
2. Add 
 
Invert to mix and store at 4 °C for up to several months; warm to 37 °C before use. The resulting cell culture media should have a final concentration of 10% or 5% (vol/vol) FBS.

 

SECTION: Reagent setup: Cell culture
3. Thaw HEK293T cells according to the manufacturer’s recommendations. Passage cells using either
TrypLE Express enzyme or a standard trypsinization protocol for adherent cultures69. Seed cells in 150-mm tissue culture dishes with a final volume of 25 mL of media per dish. Maintain in a cell culture incubator at 37 °C with 5% CO2. 

 

SECTION: Reagent setup: PEI stock solution
4. Add 323 mg of PEI to a 1 litre bottle of WFI water. PEI Once dissolved, add reserved WFI water to a total volume of 1 L. Filter-sterilize, make aliquots in 50-ml conical centrifuge tubes, and store at -20 °C for up to 1 year. We routinely freeze–thaw our PEI aliquots. 

 

SECTION: Reagent setup: PEI + DPBS master mix
5. Thaw PEI in a 37 °C water bath. Bring the PEI to Room temperature and vortex to mix. Add PEI and DPBS to a 50-ml conical centrifuge tube and vortex again to mix. Use Supplementary Table 2 from Challis et al., Nat Protoc 14: 379 (‘Transfection calculator’ sheet) to calculate the volumes of PEI (cell I9) and DPBS (cell J9) needed. 

 

SECTION: Reagent setup: DNA + DPBS
6. Bring plasmid DNA to Room temperature and briefly vortex to mix. For each viral prep, add DNA and DPBS to a 50-ml conical centrifuge tube. Use Supplementary Table 2 from Challis et al., Nat Protoc 14: 379 (‘Transfection calculator’ sheet) to calculate the quantities of DNA (e.g., cells E9, E11, and E13) and DPBS (e.g., cell F9) needed. 

 

SECTION: Reagent setup: SAN digestion buffer
7. Add 
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting SAN digestion buffer should have a final pH of ~pH 10.0 and a final concentration of 500 millimolar (mM) NaCl, 40 millimolar (mM) Tris base, and 10 millimolar (mM) MgCl2.
SECTION: Reagent setup: SAN + SAN digestion buffer
8. Add 100 U of SAN (4 µL of 25 U/µL SAN) per milliliter of SAN digestion buffer; pipette to mix.

 

SECTION: Reagent setup: 40% (wt/vol) PEG stock solution
9. Decant ~500 mL of WFI water into a 500-ml sterile bottle for later use. Add 146.1 g of NaCl to the remaining 500 mL (in the 1-liter bottle of WFI water) and shake/heat until dissolved. 
Once completely dissolved, add 400 g of PEG and heat at 37 °C 1440 min for up to 2 nights. Add reserved WFI water to a total volume of 1 L. Filter-sterilize and store at Room temperature for up to several months. The resulting stock solution should have a final concentration of 2.5 Molarity (M) NaCl and 40% (wt/vol) PEG. 

 

SECTION: Reagent setup: DPBS + high salt
10. Add
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting buffer should have a final concentration of 1 Molarity (M) NaCl, 2.5 millimolar (mM) KCl, and 1 millimolar (mM) MgCl2 (in addition to the salts in the DPBS).

SECTION: Reagent setup: DPBS + low salt
11. Add
 
and shake to mix. Filter-sterilize and store at Room temperature for up to several months. The resulting buffer should have a final concentration of 100 millimolar (mM) NaCl, 2.5 millimolar (mM) KCl, and 1 millimolar (mM) MgCl2 (in addition to the salts in the DPBS).
SECTION: Reagent setup: Iodixanol density gradient solutions (15%, 25%, 40%, and 60% (wt/vol) iodixanol)
12. For each layer, add iodixanol (OptiPrep), DPBS + high salt or DPBS + low salt, and phenol red (if
applicable) to a 50-ml conical centrifuge tube. Invert or briefly vortex to mix. Use Supplementary Table 3 from Challis et al., Nat Protoc 14: 379 to determine the volumes of each reagent needed (see note below). The 25% and 60% layers contain phenol red, which turns the solutions red and yellow, respectively, and facilitates clear demarcation of the gradient boundaries (Fig. 8).

 

SECTION: Equipment setup: Clamp setup for AAV purification
13. Attach the rod to the support stand. Secure the clamp 25–30 cm above the stand (Fig. 8f from Challis et al., Nat Protoc 14: 379).
 
 
SECTION: Procedure
14. 
 

 

SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
15. 
 

24 h before transfection, seed HEK293T cells in 150-mm dishes to attain 90% confluency the next day69. Incubate the cells in a cell culture incubator at 37 °C with 5% CO2. 

 

SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
16. At the time of transfection, make the PEI + DPBS master mix and the DNA + DPBS solution for each viral prep (Reagent setup and Supplementary Table 2 from Challis et al., Nat Protoc. 14: 379-414, ‘Transfection calculator’ sheet). Using a 5- or 10-ml serological pipette, add the required volume of the PEI + DPBS master mix (e.g., ‘Transfection calculator’ cell G9) dropwise to the DNA + DPBS solution (e.g., ‘Transfection calculator’ cells E9 + E11 + E13 + F9) while gently vortexing to mix. Cap the tube and thoroughly vortex for 10 s to mix. Allow the mixture to sit at Room temperature for 2–10 min. Add 2 mL of the transfection solution dropwise to each dish. Mix by gently pushing plate forward and backward, then side-to-side, and finally in a 'Figure 8' pattern, before returning the dishes to the cell culture incubator. 
 

SECTION: Procedure: Triple transient transfection of HEK293T cells ● Timing 1–2 h
17. Change the media 12–24 h post transfection by aspirating the old media in 10% (vol/vol) bleach and replacing it with 25 mL of fresh, pre-warmed DMEM with 5% FBS (Reagent setup).

 

SECTION: Procedure: AAV harvest ● Timing 5 d
18. 
 

 

Harvest the cell culture media 72 h (3 d) post transfection. Tilt each dish at a 30° angle and use a 25-ml serological pipette to collect the media. Store in an empty, sterile media bottle (saved from earlier expansion or media changes) 4 °C until Step 19. Replace the media with 25 mL of fresh, pre-warmed DMEM with 5% FBS (Reagent setup). While expanding the number of plates, save used media bottles. These bottles are sterile and can be used during AAV harvest (Steps 18-28).

 

SECTION: Procedure: AAV harvest ● Timing 5 d
19. Harvest the media and cells 120 h (5 d) post-transfection. Use a cell scraper to gently scrape the cells in the media. After scraping the first dish, prop it at a 30° angle, using an empty 1.5-ml microcentrifuge tube rack for support. Scrape down the residual cells and media such that they are pooled together. Return the dish lid and scrape the next plate; prop dishes up against one another along the length of the biosafety cabinet until scraping is complete. Use a 25-ml serological pipette to collect the media and cells from each dish; transfer to the 250 mL or 500 mL conical tube used during earlier media changes. Use some of the media collected at 72 h post-transfection to wash remaining cells off each plate and combine with the scraped cell suspension.
 
 

SECTION: Procedure: AAV harvest ● Timing 5 d
20. Combine the remaining media collected at 72 h post transfection with the media and cells collected at 120 h post transfection. 


 

SECTION: Procedure: AAV harvest ● Timing 5 d
21. Centrifuge the media and cells at 2000 x g, 15 min. Ensure that the tube caps are tightly secured. Centrifugation will result in the formation of a cell pellet (Fig. 7b).

 


 
SECTION: Procedure: AAV harvest ● Timing 5 d
23. Prepare 9.5 mL (if using a Type 70 Ti rotor) or 3.0 mL (if using a Type 70.1 Ti rotor) of SAN + SAN digestion buffer (Reagent setup) per viral prep. Use a 10-ml serological pipette to gently resuspend the cell pellet in 8.0 mL(Type 70 Ti) or 2.4 mL (Type 70.1 Ti) of SAN + SAN digestion buffer; pipette into a 50-ml tube to finish resuspending the pellet (Fig. 7b). Incubate in a 37 °C water bath until Step 27. Remaining SAN + SAN digestion buffer can be stored at 4 °C until Step 27. 

 

SECTION: Procedure: AAV harvest ● Timing 5 d
22. Pour off the supernatant (i.e., the clarified media) into the reserved media bottle. Centrifuge again at 2000 x g, 1 min and remove media using a P1000 pipette and add to the supernatant.

 


SECTION: Procedure: AAV harvest ● Timing 5 d
24. Retrieve the supernatant collected in Step 22, and transfer into the corresponding saved conical tube from Step 23. Using markings on the side of the conical tube, decant 1/4 of the supernatant volume of 40% (wt/vol) PEG stock solution into the supernatant (i.e. the PEG solution is formulated as 5X working concentration and the final solution should contain 8% (wt/vol) PEG). Tighten the cap and invert ten times to mix. Incubate on ice for at least 120 min. 

 

SECTION: Procedure: AAV harvest ● Timing 5 d
26. Pour off the supernatant (i.e., the PEG-clarified media) into used media collection bottle for bleaching. Centrifuge the conical tube again at 4000 x g, 5 min, 4 °C and remove remaining supernatant with a P1000 pipette and add to the already collected supernatant. 

 


SECTION: Procedure: AAV harvest ● Timing 5 d
25. Centrifuge the PEG–media mixture at 4000 x g, 45 min, 4 °C. Centrifugation will result in the formation of a PEG pellet (Fig. 7b).

SECTION: Procedure: AAV purification ● Timing 1 d
29. 
 

Determine the number of gradients needed and prepare the iodixanol density gradient solutions, if necessary (Reagent setup and Supplementary Table 3 from Challis et al., Nat Protoc. 14: 379-414). Set the OptiSeal tubes in the rack provided in the OptiSeal tube kit; alternatively, use the long edge of a 50-ml tube Styrofoam rack. 

 

SECTION: Procedure: AAV harvest ● Timing 5 d
27. PEG pellet resuspension. Resuspend the PEG pellet in 1 mL (Type 70 Ti) or 0.3 mL (Type 70.1 Ti) of SAN + SAN digestion buffer (Reagent setup) per viral prep. Using a P1000 pipette, pipette up and down to resuspend the PEG pellet until smooth and free of clumps. Combine the resuspended PEG pellet with the corresponding lysate from Step 23.

Incubate in a 37 °C water bath for an additional 30 min. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
34. Using a 2-ml serological pipette, load the remaining supernatant into the OptiSeal tube or until the volume reaches the neck of the tube. If you have loaded all the supernatant and there is still remaining space, add SAN buffer to reach the neck of the tube. 

Firmly insert a black cap (Fig. 8d from Challis et al., Nat Protoc. 14: 379-414) and place a spacer on top (Fig. 8e). Caps and spacers are provided with the OptiSeal tubes and in the OptiSeal tube kit, respectively. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
30. Pour the density gradients (Fig. 8a,b and Supplementary Video 1, 0:00–1:45, or Supplementary Video 2, 0:00–1:13 of Challis et al., Nat Protoc. 14: 379-414). Each gradient is composed of the following layers, from top-down: 

15% (wt/vol) iodixanol in high salt buffer
25% (wt/vol) iodixanol in low salt buffer, with phenol red
40% (wt/vol) iodixanol in low salt buffer
60% (wt/vol) iodixanol in low salt buffer, with phenol red. 

Pour the layers with a 2-mL serological pipette. Begin by pipetting the necessary volume of 15% (wt/vol) iodixanol to each tube. Next, add the required volume of 25% (wt/vol) iodixanol under the 15% layer by lightly touching the pipette tip to the bottom of the tube and slowly releasing the solution (Fig. 8a and Supplementary Video 1, 0:13–1:29). Continue adding layers of increasing density under the previous layer. The gradients should have a sharp delineation between layers (Fig. 8b).

For a Type 70 Ti rotor:
6 mL of 15% step
4 mL of 25% step
5 mL of 40% step
3 mL of 60% step

For a Type 70.1 Ti rotor
1.8 mL of 15% step
1.2 mL of 25% step
1.5 mL of 40% step
0.8 mL of 60% step

 

SECTION: Procedure: AAV purification ● Timing 1 d
31. Centrifuge the lysate from Step 36 at 2000 x g, 10 min. Centrifugation will result in the formation of a pellet (Fig. 7b).

 

SECTION: Procedure: AAV purification ● Timing 1 d
32. Use a 2-ml serological pipette to load the supernatant (i.e., the clarified lysate) from Step 31 above the 15% (wt/vol) iodixanol layer (Fig. 8c and Supplementary Video 1, 1:46–2:22 or Supplementary Video 2, 1:14–1:55 from Challis et al., Nat Protoc. 14: 379-414). Touch the pipette tip to the surface of the 15% layer and slowly release the lysate such that a layer forms on top.

For a Type 70 Ti rotor, you should be able to load about 9 mL  of supernatant. For a Type 70.1 Ti rotor, you should be able to load about 2.7 mL of supernatant.

 

SECTION: Procedure: AAV purification ● Timing 1 d
35. Weigh the tubes with the caps and spacers on. Balance the tubes to within 5-10 mg of each other by adding lysate or SAN digestion buffer, or by removing lysate. Be sure to adjust the tube weight in the biosafety cabinet; use the tube removal tool provided with the OptiSeal tube kit to remove the black cap and adjust the volume with a P20 or P200 pipette. The density of the SAN digestion buffer and of the lysate are close to that of water (about 1000 mg/mL )

 


SECTION: Procedure: AAV purification ● Timing 1 d
36. Load balanced tubes into the ultracentrifuge rotor and fasten the lid. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
37. Carefully transfer the rotor to the ultracentrifuge and centrifuge:

For a Type 70 Ti rotor:350000 x g, 90 min, 18 °C (58,400 r.p.m.)
For a Type 70.1 Ti rotor: 350000 x g, 75 min, 18 °C (61,700 r.p.m.)

Use slow acceleration (no. 3; the instrument will take 3 min to accelerate to 500 r.p.m., followed by maximum acceleration) and deceleration (no. 9; the instrument will deccelerate at maximum speed until it reaches 500 r.p.m., then take 6 min to stop) profiles. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
38. During ultracentrifugation, gather the supplies and equipment for Steps 39-42. Assemble the clamp setup (Equipment setup) and collect one of each of the following per gradient: PES Ultra-15 centrifugal filter device, 5-ml syringe, 10-ml syringe, 0.22-μm syringe filter unit, and a 16-gauge needle.
SECTION: Procedure: AAV purification ● Timing 1 d
39. After ultracentrifugation, bring the rotor inside the biosafety cabinet and remove the lid. Use the spacer removal tool provided with the OptiSeal tube kit to remove the spacer from the first tube. Next, use the tube removal tool to grip the tube neck. Slowly remove the tube from the rotor and secure it into the clamp setup above a 500-ml or 1-liter beaker containing fresh 10% (vol/vol) bleach (Fig. 8f of Challis et al., Nat Protoc. 14: 379-414). Clean the side of the tube with a paper towel or a Kimwipe sprayed with 70% (vol/vol) ethanol. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
40. Prepare the supplies for Steps 41 and 42. First, remove and save the plunger from a 10-ml syringe. Attach a 0.22-μm syringe filter unit to the syringe barrel and place it on top of an PES filter device. Next, add 10 mL of DPBS supplemented with 0.001% of Pluronic F-68 (DPBS + 0.001% Pluornic F-68) to the barrel and allow the solution to begin dripping through the syringe filter unit and into the filter device (Fig. 8f). Last, attach a 16-gauge needle to a 5-ml syringe. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
41. From the tube clamped in Step 46, collect the virus, starting just below the 40/60% interface, through the 40% layer9,10 (Fig. 8g and Supplementary Video 3, 0:00–1:30 from Challis et al., Nat Protoc. 14: 379-414). Hold the top of the OptiSeal tube with your nondominant hand; use your dominant hand to hold the needle/syringe. Use a forward-twisting motion to insert the needle ~4 mm below the 40/60% interface (i.e., where the tube just starts to curve). Use the tube removal tool in your non-dominant hand to remove the black cap from the tube to provide a hole for air entry. With the needle bevel up, use the needle/syringe to collect 4.0–4.5 ml of virus/ iodixanol from the 40/60% interface and 40% layer. Do not collect from the white protein layer at the 25/40% interface; as this interface is approached, rotate the needle bevel down and continue collecting from the 40% layer. Firmly replace the black cap before removing the needle from the tube. 

 

 

SECTION: Procedure: AAV purification ● Timing 1 d
42. Add the 4.0-4.5 mL of virus/iodixanol to the syringe barrel containing 10 mL of DPBS (prepared in Step 47) (Fig. 8h and Supplementary Video 3, 1:31–2:06 from Challis et al., Nat Protoc. 14: 379-414). Layer the virus below the DPBS by placing the needle near the bottom of the barrel and pressing on the plunger. Insert the 10-ml syringe plunger into the barrel and push the virus/DPBS mixture through the syringe filter unit and into the PES filter device (Supplementary Video 3, 2:07–2:32). Mix well using a P1000 pipette. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
43. Centrifuge the virus/DPBS mixture at 3000 x g for 5–8 min, or until the volume of the solution remaining in the top chamber of the PES filter device is 500-1500 µL (>10× concentrated).

 

SECTION: Procedure: AAV purification ● Timing 1 d
44. Discard the flow-through for bleaching. Add 13 mL of DPBS + 0.001% Pluornic F-68 to the virus in the top chamber and use a P1000 pipette to mix. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
45. Centrifuge the virus/DPBS mixture as in Step 50. Wash the virus two more times for a total of four buffer exchanges. During the last spin, retain 150-200 µL of solution in the top chamber. 

 


SECTION: Procedure: AAV purification ● Timing 1 d
46. Use a P200 pipette to transfer the virus from the top chamber of the PES filter device directly to a 1.6-ml screw-cap vial. Then wash the PES filter off with another 150-200 µL of DPBS + 0.001% Pluronic F-68, pipetting the wash buffer on the walls 10 times to recover any residual AAV. A second wash may help to increase yield further, but will also decrease the concentration. Combine wash(es) with the rest of the virus in the 1.6-ml screw-cap vial and store at 4 °C .

 

SECTION: Procedure: AAV harvest ● Timing 5 d
28. Store the lysate at 4 °C 5 min. 

 

SECTION: Procedure: AAV purification ● Timing 1 d
33. Re-extract AAV from the pellet, by adding 3 mL (for Type 70 Ti) or 1 mL (for Type 70.1 Ti) SAN buffer to the pellet, briefly vortexing, and centrifuging at 3000 x g, 10 min