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Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material.

par Lock, Martin;McGorray, Susan;Auricchio, Alberto;Ayuso, Eduard;Beecham, E Jeffery;Blouin, Veronique;Bosch, Fatima;Bose, Mahuya;Byrne, Barry;Caton, Tina;Chiorini, John A;Chtarto, Abdelwahed ;Clark, K Reed;Conlon, Thomas;Darmon, Christophe;Doria, Monica;Douar, Anne-Marie;Flotte, Terence R;Francis, Joyce;Francois, Achille;Giacca, Mauro;Korn, Michael;Korytov, Irina;Leon, Xavier;Leuchs, Barbara;Lux, Gabriele;Melas, Catherine;Mizukami, Hiroaki;Moullier, Philippe;Muller, M;Ozawa, Keiya;Philipsberg, Tina;Poulard, Karine;Raupp, Christina;Riviere, Christelle;Roosendaal, Sigrid;Samulski, R Jude;Soltys, Steven;Surosky, R T;Tenenbaum, Liliane ;Thomas, Darby L;van Montfort, Bart;Veres, Gabor;Wright, J Fraser;Xu, Yili;Zelenaia, Olga;Zentilin, Lorena;Snyder, Richard O
Référence Human gene therapy
Publication Publié, 2010-05

Article révisé par les pairs

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Résumé :

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 using 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 sixteen laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ( : 9.18 x 1011 particles/ml; 95% CI : 7.89 x 1011 to 1.05 x 1012 particles/mL), vector genomes ( : 3.28 x 1010 vg/ml; 95% CI : 2.70 x 1010 to 4.75 x 1010 vg/ml), transducing units ( :5.09 x 108 TU/ml; 95% CI :2.00 x 108 to 9.60 x 108 TU/ml) and infectious units ( : 4.37 x 109 TCID50IU/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 non-vector 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 inter-laboratory 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 Tissue Type Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards.