Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material
Authors
Lock, MartinMcGorray, Susan
Auricchio, Alberto
Ayuso, Eduard
Beecham, E. Jeffrey
Blouin-Tavel, Veronique
Bosch, Fatima
Bose, Mahuya
Bryne, Barry J.
Caton, Tina
Chiorini, John A.
Chtarto, Abdelwahed
Clark, K. Reed
Conlon, Thomas
Darmon, Christophe
Doria, Monica
Douar, Anne
Flotte, Terence R.
Francis, Joyce D.
Francois, Achille
Giacca, Mauro
Korn, Michael T.
Korytov, Irina
Leon, Xavier
Leuchs, Barbara
Lux, Gabriele
Melas, Catherine
Mizukami, Hiroaki
Moullier, Philippe
Muller, Marcus
Ozawa, Keiya
Philipsberg, Tina
Poulard, Karine
Raupp, Christina
Riviere, Christel
Roosendaal, Sigrid D.
Samulski, Richard Jude
Soltys, Steven M.
Surosky, Richard
Tenenbaum, Liliane
Thomas, Darby L.
van Montfort, Bart
Veres, Gabor
Wright, J. Fraser
Xu, Yili
Zelenaia, Olga
Zentilin, Lorena
Snyder, Richard O.
Document Type
Journal ArticlePublication Date
2010-10-14Keywords
Biological AssayDNA, Viral
*Dependovirus
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
*Genetic Vectors
Genome, Viral
Helper Viruses
Polymerase Chain Reaction
Reference Standards
Transduction, Genetic
Virus Replication
Allergy and Immunology
Genetics and Genomics
Pediatrics
Metadata
Show full item recordAbstract
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 10(1)(1) particles/ml; 95% confidence interval [CI], 7.89 x 10(1)(1) to 1.05 x 10(1)(2) particles/ml), vector genomes ({X}, 3.28 x 10(1) vector genomes/ml; 95% CI, 2.70 x 10(1) to 4.75 x 10(1) 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.Source
Hum Gene Ther. 2010 Oct;21(10):1273-85. Link to article on publisher's siteDOI
10.1089/hum.2009.223Permanent Link to this Item
http://hdl.handle.net/20.500.14038/43839PubMed ID
20486768Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1089/hum.2009.223