Rapid and simplified purification of recombinant adeno-associated virus

Preclinical gene therapy studies both in vitro and in vivo require high purity preparations of adeno-associated virus (AAV). Current methods for purification of AAV entail the use of centrifugation over either a CsCl or iodixanol gradient, or the use of chromatography. These methods can be cumbersome and expensive, necessitating ultrahigh speed gradient centrifugation or, for chromatography the use of other expensive equipment. In addition, these methods are time consuming, and the viral yield is not high. Currently no commercial purification kits are available for other than AAV serotype 2. A simplified method was used for the purification of AAV, with a viral yield that is able to be used effectively in adult and embryo mice. The method does not require ultrahigh speed gradient centrifugation nor chromatography. Instead, polyethylene glycol (PEG)/aqueous two-phase partitioning is used to remove soluble proteins from the PEG8000 precipitated virus–protein mixture. The procedure obtained rapidly up to 95% recovery of high quality purified AAV. The entire purification process, including HEK293 cell transfection, can be completed readily within one week, with purity seemingly higher than that obtained after one round of CsCl gradient purification.

► The optimal condition for aqueous two-phase partitioning to purify AAV, i.e. 10%PEG8000–13.2 (NH4)2SO4 at pH 8.0 in HEPES buffer, yielded a purity even higher than conventional CsCl gradient density centrifugation methods, and a higher efficiency of infection in vivo.
► In addition, the infection in embryonic mouse organs demonstrated that the PEG8000/(NH4)2SO4 aqueous two-phase partitioning purified AAV is not toxic to the embryos, and is sufficiently pure for in vivo studies in mouse embryos.
► The method does not require ultrahigh speed gradient centrifugation nor chromatography.