For each batch of rAAV vector, HEK293 cells are seeded into 5 × 15 cm diameter tissue culture plates in 25 mL complete DMEM. The number of cells in one fully confluent T175 flask is sufficient to seed 3.5–3.75 15 cm plates, such that the plates are 70 % confluent at the time of transfection (see Notes 2 and 3).

The following day, 3 h prior to transfection, remove the media from each plate and replace with 25 mL IMDM.

Bring transfection reagents to room temperature, and immediately prior to transfection, prepare the DNA mix in a 50 mL tube as follows:

Sterilize the DNA mix by filtration through a 0.2 μm Acrodisc syringe filter into a 75 cm² tissue culture flask.

For the transfection, whilst vortexing the DNA mix vigorously, add 13 mL of 2× HeBS buffer. The combined mixture is vortexed for a further 10–15 s and then left to stand at room temperature for a further 1 min and 45 s (see Notes 5 and 6).

Gently pipette 5 mL of the transfection mix into each 15 cm plate of HEK293 cells in IMDM in a circular dropwise motion. Agitate the plates to ensure adequate distribution of transfection mix and return the plates to the tissue culture incubator.

Sixteen hours later, remove the media and replace with 25 mL fresh prewarmed (37 °C) complete DMEM and return plates to the incubator.

Sixty hours after transfection, harvest the HEK293 cells as follows: remove the media and gently pipette ~25 mL prewarmed 1× PBS, swirl the plate gently, and remove the PBS and discard. Add 25 mL 1× PBS and detach the cells from the plate using a cell scraper and collect in sterile 50 mL tubes.

The cells are pelleted by centrifugation at 600 × g for 30 min in a centrifuge equipped with a swing-out rotor. Discard the supernatant and proceed to Sects. 3.1.3 or 3.1.4.

From Sect. 3.1.2, resuspend the cell pellets in 50 mL 150 mM NaCl, 20 mM Tris–HCl pH 8.0. Split into two 25 mL aliquots in 50 mL tubes, and store overnight at −20 °C or continue on to step 2.

Thaw the two 25 mL aliquots of cell lysate, and add sodium deoxycholate to a final concentration of 0.5 % (1.25 mL of 10 % (w/v) sodium deoxycholate) and Benzonase to a final concentration of 50 U/mL.

After mixing thoroughly, incubate the tubes in a 37 °C water bath for 1 h. Vortex the tubes every 15 min during the incubation period.

Centrifuge the tubes at 3,000 × g for 15 min, and pool the supernatants into a fresh 50 mL tube before storing overnight at −20 °C prior to heparin column purification.

Thaw the supernatants and centrifuge at 4,000 × g for 15 min to remove any protein precipitate.

Pre-equilibrate a 1 mL Hi-Trap heparin HP column with 15 mL 150 mM NaCl, 20 mM Tris–HCl pH 8.0, using a Harvard infusion pump at a flow rate of 1 mL/min and a disposable 60 mL syringe. Load the cell supernatant into the 60 mL syringe and load the sample onto the heparin column.

Wash the column with 30 mL 100 mM NaCl, 20 mM Tris–HCl pH 8.0.

Remove the column from the infusion pump and manually elute from the column into separate tubes at a rate of 1 mL/min using 1–5 mL disposable syringes with the following buffers:

Pool the 400–500 mM NaCl, 20 mM Tris–HCl pH 8.0 fractions, and transfer to a 4 mL 100K MWCO Amicon centrifugal concentrator.

Centrifuge at 3,400 × g at 4 °C to a volume of ~100–200 μL. Add 4 mL 1× PBS-MK containing 0.001 % pluronic acid to the concentrator, and centrifuge sample to a volume of ~100–200 μL. Perform a second wash step with 4 mL 1× PBS-MK containing 0.001 % pluronic acid, and concentrate the sample to a volume of ~150 μL.

Transfer the sample to a sterile 1.5 mL tube. To ensure all the rAAV is retrieved, rinse the concentrator with a further 150–200 μL of 1× PBS-MK, and pool this sample with the first 150 μL sample.

The rAAV vector stock (~350 μL) is then filter sterilized through a 13 mm 0.2 μm low-protein binding Acrodisc filter into a fresh sterile 1.5 mL tube (final volume ~300 μL).

Aliquot the vector and store at −80 °C.

From Sect. 3.1.2, resuspend the cell pellet to a final volume of 8 mL in lysis buffer (150 mM NaCl, 50 mM Tris–HCl pH 8.5). The lysate can be frozen at this stage or following the next deoxycholate/Benzonase step.

Add sodium deoxycholate to a final concentration of 0.5 % (400 μL of 10 % (w/v) sodium deoxycholate in 8 mL) and Benzonase to a final concentration of 50 U/mL. Mix well and incubate in a 37 °C water bath for 60 min. Vortex the tubes every 15 min during the incubation period.

Centrifuge the lysate at 3,000 × g for 30 min using a fixed angle rotor. Transfer the supernatant into a clean tube and freeze samples at −20 °C.

The following day, thaw cell lysates and centrifuge at 5,000 × g for 30 min before transferring the supernatant to a fresh tube.

Set up the gradient in a disposable 35 mL ultracentrifuge tube. We use Sorvall Ultracrimp tubes and sealing system. Load the gradient in the following order using a spinal needle attached to a 10 mL disposable syringe. Hold the needle against the side of the tube and slowly expel each solution so as to not disturb the layer above.

Very carefully add lysis buffer to the cell lysate (at the top of the tube) to within 1–2 mm below the bottom of the plastic stopper. Weigh each tube and lid (plastic stopper and cap) and tube adapter.

Generate another tube in a similar manner with another sample or lysis buffer to act as a balance for ultracentrifugation. Once pairs of tubes are generated, insert stopper, metal cap, and crimp the tube. Place a pen mark at the 40–54 % iodixanol interface to aid identification for sample retrieval following centrifugation.

Place tubes in Sorvall ultracentrifuge T865 rotor and centrifuge at 58,000 rpm for 1 h and 45 min. This will include a 15 min acceleration and 90 min spin. Remove tubes carefully from rotor.

In a tissue culture hood, place the tube in a clamp stand. Insert an 18 G needle attached to a 5 mL syringe at the 40–54 % iodixanol interface. Insert a 21 G needle at the top of the tube to facilitate entry of air into the tube. With the bevel of the needle facing upwards, carefully draw off 3.5 mL of the 40 % iodixanol layer containing vector and transfer this to a 50 mL tube.

Add 10 mL of 1× PBS-MK containing 0.001 % pluronic acid to the vector sample, and load into a 15 mL 100 kDa molecular weight cut-off Amicon concentrator. Centrifuge at 3,400 × g until the volume is reduced to ~200 μL. Add 14–15 mL 1× PBS-MK with pluronic acid and centrifuge as above. Repeat 2–3 times until the iodixanol is removed. When adding the wash buffer, gently pipette the sample up and down to determine whether iodixanol is present.

On the final wash, centrifuge to approximately 100 μL volume and remove the vector into a 1.5 mL tube. Add 250 μL of wash buffer to the concentrator, rinse the inside, and combine with the first sample.