title: Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
abstract: Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
authors.affiliation: 1 Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology, School of Veterinary Medicine, Universitat Autònoma de Barcelona , 08193-Bellaterra, Spain .
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authors.lastname: Ayuso
authors.forename: Véronique
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authors.lastname: Blouin
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authors.forename: Christophe
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authors.lastname: Kleinschmidt
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authors.lastname: Leuchs
authors.forename: Catherine
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authors.lastname: Melas
authors.forename: Hiroaki
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authors.lastname: Mizukami
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authors.lastname: Müller
authors.forename: Yvet
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authors.lastname: Noordman
authors.forename: Olivier
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authors.lastname: Bockstael
authors.forename: Keiya
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authors.lastname: Ozawa
authors.forename: Catherine
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authors.forename: Marina
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authors.lastname: Sumaroka
authors.forename: Richard
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authors.lastname: Surosky
authors.forename: Liliane
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authors.lastname: Tenenbaum
authors.forename: Inge
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authors.lastname: van der Linden
authors.forename: Brigitte
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authors.forename: J Fraser
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authors.lastname: Wright
authors.forename: Xinhua
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authors.lastname: Zhang
authors.forename: Lorena
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authors.lastname: Zentilin
authors.forename: Fatima
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authors.lastname: Bosch
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mesh_terms: D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D012015:Reference Standards; D014170:Transformation, Genetic; D014771:Virion / Q000235:genetics; D014776:Virus Cultivation / Q000592:standards