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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.


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?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?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? 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.

SUBMITTER: Lock M 

PROVIDER: S-EPMC2957240 | biostudies-other | 2010 Oct

REPOSITORIES: biostudies-other

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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.

Lock Martin M   McGorray Susan S   Auricchio Alberto A   Ayuso Eduard E   Beecham E Jeffrey EJ   Blouin-Tavel Véronique V   Bosch Fatima F   Bose Mahuya M   Byrne Barry J BJ   Caton Tina T   Chiorini John A JA   Chtarto Abdelwahed A   Clark K Reed KR   Conlon Thomas T   Darmon Christophe C   Doria Monica M   Douar Anne A   Flotte Terence R TR   Francis Joyce D JD   Francois Achille A   Giacca Mauro M   Korn Michael T MT   Korytov Irina I   Leon Xavier X   Leuchs Barbara B   Lux Gabriele G   Melas Catherine C   Mizukami Hiroaki H   Moullier Philippe P   Müller Marcus M   Ozawa Keiya K   Philipsberg Tina T   Poulard Karine K   Raupp Christina C   Rivière Christel C   Roosendaal Sigrid D SD   Samulski R Jude RJ   Soltys Steven M SM   Surosky Richard R   Tenenbaum Liliane L   Thomas Darby L DL   van Montfort Bart B   Veres Gabor G   Wright J Fraser JF   Xu Yili Y   Zelenaia Olga O   Zentilin Lorena L   Snyder Richard O RO  

Human gene therapy 20101001 10


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 contaminat  ...[more]

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