[48] Production and purification of recombinant adeno-associated virus

Recombinant adeno-associated virus (rAAV), because of its simplicity, ability to infect a wide variety of dividing and nondividing cells, and lack of human pathogenicity, has proved to be a useful vector for efficient and long-term gene transfer in vivo. The traditional rAAV purification protocol involved the stepwise precipitation of rAAV with ammonium sulfate, followed by two or three rounds of CsC1 density gradient centrifugation. Each gradient required fractionation and identification of the virus-containing regions by dot-blot hybridization or by polymerase chain reaction (PCR) analysis. This chapter presents a protocol that substantially reduces preparation time without sacrificing the quality and purity of the final product. It is based on two improvements: (1) the observation that AAV binds to cell surface heparin sulfate proteoglycan, and (2) a new bulk purification technique employing the nonionic gradient medium, iodixanol, which allows efficient binding of the virus to the affinity medium. This combination of techniques results in high recovery rates, improved viral infectivity, and rapid purification.