title: Insect cells as a factory to produce adeno-associated virus type 2 vectors.
abstract: Recombinant adeno-associated viruses (rAAV) are produced transiently in mammalian cells usually by cotransfecting two or three plasmids containing AAV genes, adenovirus helper genes, and a vector genome. Expansion and transfection of adherent cells limit the scale of rAAV production. Efficient transfection is performed with cells on solid support media such as tissue culture plates. A large animal study or a human clinical trial may require 10(15) particles of vector, depending on dose. To generate this quantity of rAAV by transfection, more than 10(11) HEK293 cells may be needed, which would require about 5000 x 175 cm(2) flasks. The ability to scale up rAAV production by these methods severely restricts the commercialization and use of AAV vectors. A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus is widely employed for large-scale production of heterologous proteins in cultured insect cells and may provide an attractive alternative. Toward this goal, we have explored the production of rAAV in invertebrate cells. Sf9 cells may be coinfected in suspension cultures with three recombinant baculoviruses (a Rep-baculovirus, a VP-baculovirus, and an AAV ITR vector genome baculovirus) and, 3 days later, rAAV is recovered. The particles produced are indistinguishable from 293 cell-produced rAAV, as determined on the basis of physical properties and biologic activities. Particles produced by either method were composed of similar proteins and nucleic acid. The yield of genome-containing particles produced per Sf9 cell approached 5 x 10(4), thus, 1000 ml of cultured Sf9 cells produced the equivalent of between 500 to 1000 x 175 cm(2) flasks of 293 cells. This robust system provides a simple, cost-effective method for AAV vector production.
authors.affiliation: Laboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Masashi
authors.initials: M
authors.lastname: Urabe
authors.forename: Chuantian
authors.initials: C
authors.lastname: Ding
authors.forename: Robert M
authors.initials: RM
authors.lastname: Kotin
mesh_terms: D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D000229:Dependovirus / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007313:Insecta; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D002712:Chlorides; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002586:Cesium; C028019:cesium chloride