title: Small-Scale Recombinant Adeno-Associated Virus Purification.
abstract: Recombinant adeno-associated virus (rAAV) vectors have become increasingly popular in research and clinical trials due to their efficient gene transfer and long-term expression in tissues including brain. In addition, rAAV has demonstrated an impressive safety profile in gene therapy trials. The emergence of rAAV serotypes with different cell tropisms and distribution properties has allowed scientists to tailor serotypes to specific experimental needs. AAV does not have a cytopathic effect; therefore, purification methods require extraction of the viral vector from the cell. This involves gradient ultracentrifugation of the cellular extract sometimes followed by chromatography. This chapter describes a small-scale production method for rAAV purification from ten to twenty 15 cm plates of human embryonic kidney-derived 293B cells (HEK 293) cells that can yield approximately 300 μl of a 5 × 10(12) to 1 × 10(13) genome copies/ml viral preparation final concentration.
authors.affiliation: Department of Neurology, Medical Sciences Center, University of Wisconsin-Madison, 1300 University Avenue, Room 73 Bardeen, Madison, WI, 53706, USA. burger@neurology.wisc.edu.
authors.forename: Corinna
authors.initials: C
authors.lastname: Burger
authors.affiliation: Molecular Pharmacology and Physiology, Byrd Alzheimer Institute, College of Medicine, University of South Florida, 4001 E. Fletcher Avenue, MDC Box 36, Tampa, FL, 33612, USA.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Nash
mesh_terms: D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification*; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D014283:Triiodobenzoic Acids / Q000737:chemistry*; D019562:Viral Load
chemical_list: D014283:Triiodobenzoic Acids; C044834:iodixanol
keywords: Adeno-associated virus; Iodixanol gradient purification; Tissue culture