A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction

Recombinant adeno-associated virus (AAV) is becoming the vector of choice f or many gene therapy protocols. There has been much recent progress made to ward increasing AAV titres but a continuing problem in using AAV has been t hat it is relatively difficult to concentrate and purify. Traditional metho ds, such as caesium chloride (CsCl) gradients, have drawbacks, notably exte nded purification times and the ability to process only limited volumes. Wh ere the target cells of interest require a high multiplicity of infection ( MOI), or to complete in vivo experiments, there is a requirement for both t he production of high titre and a large volume of virus. This is laborious to obtain using traditional methods. A simple technique is described here f or purifying AAV, involving affinity chromatography, protease digestion and solvent extraction that retains both the high yields and titres obtained u sing CsCl gradients. In addition, this technique displays a fast throughput and may be used to purify AAV from larger volumes than CsCl gradients. The high yield and purity of these virus preparations has allowed us to achiev e good levels of expression in the target cell types tested. The purificati on technique described here will be applicable to any protocol that require s high titre, high purity recombinant AAV (rAAV). (C) 2000 Elsevier Science B.V. All rights reserved.