Characterization of a recombinant adeno-associated virus type 2 reference standard material

Martin Lock, Susan McGorray, Alberto Auricchio, Eduard Ayuso, E. Jeffrey Beecham, Véronique Blouin-Tavel, Fatima Bosch, Mahuya Bose, Barry J. Byrne, Tina Caton, John A. Chiorini, Abdelwahed Chtarto, K. Reed Clark, Thomas Conlon, Christophe Darmon, Monica Doria, Anne Douar, Terence R. Flotte, Joyce D. Francis, Achille FrancoisMauro Giacca, Michael T. Korn, Irina Korytov, Xavier Leon, Barbara Leuchs, Gabriele Lux, Catherine Melas, Hiroaki Mizukami, Philippe Moullier, Marcus Müller, Keiya Ozawa, Tina Philipsberg, Karine Poulard, Christina Raupp, Christel Rivière, Sigrid D. Roosendaal, R. Jude Samulski, Steven M. Soltys, Richard Surosky, Liliane Tenenbaum, Darby L. Thomas, Bart Van Montfort, Gabor Veres, J. Fraser Wright, Yili Xu, Olga Zelenaia, Lorena Zentilin, Richard O. Snyder

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82 Citations (Scopus)

Abstract

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18×1011 particles/ml; 95% confidence interval [CI], 7.89×1011 to 1.05×1012 particles/ml), vector genomes ({X}, 3.28×1010 vector genomes/ml; 95% CI, 2.70×1010 to 4.75×1010 vector genomes/ml), transducing units ({X}, 5.09×108 transducing units/ml; 95% CI, 2.00×108 to 9.60×108 transducing units/ml), and infectious units ({X}, 4.37×109 TCID50 IU/ml; 95% CI, 2.06×109 to 9.26×109 TCID50 IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed. © 2010 Mary Ann Liebert, Inc.
Original languageEnglish
Pages (from-to)1273-1285
JournalHuman Gene Therapy
Volume21
DOIs
Publication statusPublished - 1 Oct 2010

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