title: Rapid and simplified purification of recombinant adeno-associated virus.
abstract: 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.
authors.affiliation: Department of Surgery, Division of Pediatric Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh, PA 15224, USA. ping.guo@chp.edu
authors.forename: Ping
authors.initials: P
authors.lastname: Guo
authors.forename: Yousef
authors.initials: Y
authors.lastname: El-Gohary
authors.forename: Krishna
authors.initials: K
authors.lastname: Prasadan
authors.forename: Chiyo
authors.initials: C
authors.lastname: Shiota
authors.forename: Xiangwei
authors.initials: X
authors.lastname: Xiao
authors.forename: John
authors.initials: J
authors.lastname: Wiersch
authors.forename: Jose
authors.initials: J
authors.lastname: Paredes
authors.forename: Sidhartha
authors.initials: S
authors.lastname: Tulachan
authors.forename: George K
authors.initials: GK
authors.lastname: Gittes
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D002021:Buffers; D002254:Carbonates / Q000737:chemistry; D011232:Chemical Precipitation; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D059625:Liquid-Liquid Extraction; D008099:Liver / Q000378:metabolism / Q000821:virology; D051379:Mice; D030781:Organisms, Genetically Modified; D011092:Polyethylene Glycols / Q000737:chemistry; D012462:Saline Solution, Hypertonic / Q000737:chemistry; D014161:Transduction, Genetic
chemical_list: D002021:Buffers; D002254:Carbonates; D012462:Saline Solution, Hypertonic; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; C005686:sodium carbonate; D000645:Ammonium Sulfate