Small-scale rAAV 1, 2, and 5 vector purification

Cells are lysed by three freeze/thaw cycles between dry ice-ethanol and 37 °C water baths. Benzonase (Sigma) is then added to the cell lysate (50 U/ml final concentration) and incubated for 30 min at 37 °C. The crude lysate is clarified by centrifugation at 4000 x g for 20 min and the vector-containing supernatant is divided between four iodixanol gradients. 

Discontinuous iodixanol step gradients are formed in quick seal tubes (25 x 89 mm, Beckman) by underlaying and displacing the less dense cell lysate (15 ml) with iodixanol (5,5 [(2-hydroxy-1-3-propanediyl)-bis(acetylamino)] bis [N,Ar,-bis(2,3dihydroxypro-pyl-2,4,6-triiodo-l,3-benzenecarboxamide]) prepared using a 60% (w/v) sterile solution of OptiPrep (Nycomed) and PBS-MK buffer (1 x PBS containing 1 mM MgCl2 and 2.5 mM KC1). Therefore, 

For rAAVl and 5 vectors, a 5 ml HiTrap Q column (Pharmacia) is equilibrated at 5 ml/min with 5 column volumes (25 ml) of Buffer A (20 mM Tris, 15 mM NaCl, pH 8.5), then 25 ml Buffer B (20 mM Tris, 500 mM NaCl, pH 8.5), followed by 25 ml of Buffer A using a Pharmacia ATKA FPLC system. The 20 ml vector-containing iodixanol fraction is diluted 1 1 with Buffer A and applied to the column at a flow rate of 3-5 ml/min. After loading the sample, the column is washed with 10 column volumes (50 ml) of Buffer A. The vector is eluted with Buffer B and fractions are collected. 

ADENO-ASSOCIATED VIRUS VECTORS FOR GENE THERAPY 

If necessary, the vector can be concentrated, and desalted or buffer exchanged in a Biomax 100 K concentrator (Millipore) by three cycles of centrifugation. In each cycle the virus is concentrated to 1 ml following the addition of 10 ml of Lactated Ringer s or 1 x PBS. The virus is stored at -80 °C in lactated Ringer s or 1 x PBS. 

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