title: A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells.
abstract: Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.
authors.affiliation: Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Smith
authors.forename: Justin R
authors.initials: JR
authors.lastname: Levy
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kotin
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D000229:Dependovirus / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D018411:Spodoptera