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

Human gene therapy (2014-10-03)
Eduard Ayuso, Véronique Blouin, Martin Lock, Susan McGorray, Xavier Leon, Mauricio R Alvira, Alberto Auricchio, Stephanie Bucher, Abdelwahed Chtarto, K Reed Clark, Christophe Darmon, Monica Doria, Will Fountain, Guangping Gao, Kai Gao, Mauro Giacca, Juergen Kleinschmidt, Barbara Leuchs, Catherine Melas, Hiroaki Mizukami, Marcus Müller, Yvet Noordman, Olivier Bockstael, Keiya Ozawa, Catherine Pythoud, Marina Sumaroka, Richard Surosky, Liliane Tenenbaum, Inge van der Linden, Brigitte Weins, J Fraser Wright, Xinhua Zhang, Lorena Zentilin, Fatima Bosch, Richard O Snyder, Philippe Moullier
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Ampicillin, anhydrous, 96.0-102.0% (anhydrous basis)
Sigma-Aldrich
Phenylacetic acid, 99%
Supelco
Ampicillin, analytical standard
Sigma-Aldrich
Ampicillin, meets USP testing specifications
USP
Ampicillin, United States Pharmacopeia (USP) Reference Standard
Ampicillin, anhydrous, European Pharmacopoeia (EP) Reference Standard