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 for many gene therapy protocols. There has been much recent progress made toward increasing AAV titres but a continuing problem in using AAV has been that it is relatively difficult to concentrate and purify. Traditional methods, such as caesium chloride (CsCl) gradients, have drawbacks, notably extended purification times and the ability to process only limited volumes. Where the target cells of interest require a high multiplicity of infection (MOI), or to complete in vivo experiments, there is a requirement for both the production of high titre and a large volume of virus. This is laborious to obtain using traditional methods. A simple technique is described here for purifying AAV, involving affinity chromatography, protease digestion and solvent extraction that retains both the high yields and titres obtained using 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 achieve good levels of expression in the target cell types tested. The purification technique described here will be applicable to any protocol that requires high titre, high purity recombinant AAV (rAAV).
Anderson
R
R
Department of Haematology, Royal Free and University College Medical School of University College London, UK. r.anderson@rfc.ucl.ac.uk
Macdonald
I
I
Corbett
T
T
Whiteway
A
A
Prentice
H G
HG
D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D000229:Dependovirus / Q000302:isolation & purification*; D010450:Endopeptidases; D005822:Genetic Vectors; D006801:Humans; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012997:Solvents
D012997:Solvents; D010450:Endopeptidases