Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material

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,...

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Published in:	Human gene therapy Vol. 25; no. 11; p. 977
Main Authors:	Ayuso, Eduard, Blouin, Véronique, Lock, Martin, McGorray, Susan, Leon, Xavier, Alvira, Mauricio R, Auricchio, Alberto, Bucher, Stephanie, Chtarto, Abdelwahed, Clark, K Reed, Darmon, Christophe, Doria, Monica, Fountain, Will, Gao, Guangping, Gao, Kai, Giacca, Mauro, Kleinschmidt, Juergen, Leuchs, Barbara, Melas, Catherine, Mizukami, Hiroaki, Müller, Marcus, Noordman, Yvet, Bockstael, Olivier, Ozawa, Keiya, Pythoud, Catherine, Sumaroka, Marina, Surosky, Richard, Tenenbaum, Liliane, van der Linden, Inge, Weins, Brigitte, Wright, J Fraser, Zhang, Xinhua, Zentilin, Lorena, Bosch, Fatima, Snyder, Richard O, Moullier, Philippe
Format:	Journal Article
Language:	English
Published:	United States 01-11-2014
Subjects:	Dependovirus - genetics Genetic Therapy Genome, Viral HEK293 Cells Humans Reference Standards Transformation, Genetic Virion - genetics Virus Cultivation - standards
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Summary:	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.
ISSN:	1557-7422
DOI:	10.1089/hum.2014.057