Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 140

5. Thaw the supernatants and centrifuge at 4,000 × g for 15 min
to remove any protein precipitate.
6. 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 dis-
posable 60 mL syringe. Load the cell supernatant into the
60 mL syringe and load the sample onto the heparin column.
7. Wash the column with 30 mL 100 mM NaCl, 20 mM Tris-
HCl pH 8.0.
8. 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:
1 mL 200 mM NaCl, 20 mM Tris-HCl pH 8.0
1 mL 300 mM NaCl, 20 mM Tris-HCl pH 8.0
1.5 mL 400 mM NaCl, 20 mM Tris-HCl pH 8.0
3 mL 450 mM NaCl, 20 mM Tris-HCl pH 8.0
1.5 mL 500 mM NaCl, 20 mM Tris-HCl pH 8.0
9. Pool the 400-500 mM NaCl, 20 mM Tris-HCl pH 8.0 frac-
tions, and transfer to a 4 mL 100K MWCO Amicon centrifu-
gal concentrator.
10. 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 concen-
trate the sample to a volume of ~150 μL.
11. 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.
12. 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).
13. Aliquot the vector and store at −80 °C.

This method is suitable for the purification for all AAV serotypes
and as an alternative purification method for serotype 2 or chimeric
AAV1:2 vectors:

3.1.4 Iodixanol
Purification Method for All
AAV Serotypes

1. 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.