Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material
| Autor: | Alberto Auricchio, Christel Rivière, Michael Korn, Karine Poulard, R. Jude Samulski, Thomas J. Conlon, Mahuya Bose, Steven M. Soltys, Richard O. Snyder, John A. Chiorini, Irina Korytov, Gabriele Lux, Monica Doria, Achille François, Sigrid D. Roosendaal, Christina Raupp, Bart van Montfort, Hiroaki Mizukami, Darby L. Thomas, Fatima Bosch, Tina Caton, J. Fraser Wright, E. Jeffrey Beecham, Abdelwahed Chtarto, Olga Zelenaia, Gabor Veres, Martin Lock, Véronique Blouin-Tavel, Christophe Darmon, Xavier León, Lorena Zentilin, K. Reed Clark, Mauro Giacca, Liliane Tenenbaum, Richard T. Surosky, Keiya Ozawa, Joyce D. Francis, Terence R. Flotte, Tina Philipsberg, Philippe Moullier, Barry J. Byrne, Eduard Ayuso, Catherine Melas, Barbara Leuchs, Susan P. McGorray, Marcus Müller, Anne Douar, Yili Xu |
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| Přispěvatelé: | M., Lock, S., Mcgorray, A., Auricchio, E., Ayuso, E. J., Beecham, V., Blouin Tavel, F., Bosch, M., Bose, B. J., Byrne, T., Caton, J. A., Chiorini, A., Chtarto, K. R., Clark, T., Conlon, C., Darmon, M., Doria, A., Douar, T. R., Flotte, J. D., Franci, A., Francoi, Giacca, Mauro, M. T., Korn, I., Korytov, X., Leon, B., Leuch, G., Lux, C., Mela, H., Mizukami, P., Moullier, M., Müller, K., Ozawa, T., Philipsberg, K., Poulard, C., Raupp, C., Rivière, S. D., Roosendaal, R. J., Samulski, S. M., Solty, R., Surosky, L., Tenenbaum, D. L., Thoma, B. v., Montfort, G., Vere, J. F., Wright, Y., Xu, O., Zelenaia, L., Zentilin, R. O., Snyder, Auricchio, Alberto, V., Blouin, B., Byrne, J., Chiorini, A. M., Douar, J., Franci, M., Giacca, M., Korn, M., Muller, C., Riviere, S., Roosendaal, S., Solty, B., van Montfort |
| Jazyk: | angličtina |
| Rok vydání: | 2010 |
| Předmět: |
Serotype
Electrophoresis Genetic Virus Replication viruses Genetic Vectors isolation /&/ purification Enzyme-Linked Immunosorbent Assay Genome Viral Biology Viral Helper Viruses Polymerase Chain Reaction Reference Standards Transduction Recombinant virus chemistry Virus Replication Polymerase Chain Reaction law.invention Transduction isolation /&/ purification Genome Genetic law Transduction Genetic Genetics Viral Adeno-Associated Virus Type 2 Molecular Biology Research Articles Biological Assay DNA chemistry Dependovirus classification/genetics/isolation /&/ purification/physiology Electrophoresis Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay Genetic Vectors Polyacrylamide Gel Genome classification/genetics/isolation /&/ purification/physiology Electrophoresi DNA Dependovirus Reference Standards Molecular biology Confidence interval Titer Capsid Helper virus DNA Viral chemistry Dependoviru Recombinant DNA Molecular Medicine Electrophoresis Polyacrylamide Gel Biological Assay classification/genetics/isolation /&/ purification/physiology Helper Viruses Polyacrylamide Gel Enzyme-Linked Immunosorbent Assay Genetic Vector |
| Popis: | 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 x 10¹¹ particles/ml; 95\% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95\% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95\% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95\% CI, 2.06 x 10⁹ to 9.26 x 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. |
| Databáze: | OpenAIRE |
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