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


Martin Lock, University of Pennsylvania
Susan McGorray, University of Florida College of Medicine
Alberto Auricchio, Federico II University
Eduard Ayuso, Universitat Autònoma de Barcelona
E. Jeffrey Beecham, University of North Carolina
Veronique Blouin-Tavel, Centre Hospitalier Universitaire de Nantes
Fatima Bosch, Universitat Autònoma de Barcelona
Mahuya Bose, University of Florida
Barry J. Bryne, University of Florida
Tina Caton, University of Florida
John A. Chiorini, National Institutes of Health
Abdelwahed Chtarto, Free University of Brussels
K. Reed Clark, Ohio State University
Thomas Conlon, University of Florida
Christophe Darmon, Centre Hospitalier Universitaire de Nantes
Monica Doria, Telethon Institute of Genetics and Medicine
Anne Douar, Généthon
Terence R. Flotte, University of Massachusetts Medical SchoolFollow
Joyce D. Francis, University of Florida
Achille Francois, Centre Hospitalier Universitaire de Nantes
Mauro Giacca, International Center for Genetic Engineering and Biotechnology
Michael T. Korn, University of Pennsylvania
Irina Korytov, University of Florida
Xavier Leon, Universitat Autònoma de Barcelona
Barbara Leuchs, German Cancer Research Center
Gabriele Lux, Pharmazeutische Fabrik Dr. Reckeweg
Catherine Melas, Free University of Brussels
Hiroaki Mizukami, Jichi Medical University
Philippe Moullier, Centre Hospitalier Universitaire de Nantes
Marcus Muller, German Cancer Research Center
Keiya Ozawa, Jichi Medical University
Tina Philipsberg, University of Florida
Karine Poulard, Généthon
Christina Raupp, German Cancer Research Center
Christel Riviere, Généthon
Sigrid D. Roosendaal, Amsterdam Molecular Therapeutics
Richard Jude Samulski, University of Florida
Steven M. Soltys, University of North Carolina
Richard Surosky, Sangamo BioSciences
Liliane Tenenbaum, Free University of Brussels
Darby L. Thomas, Applied Genetic Technologies Corporation
Bart van Montfort, Amsterdam Molecular Therapeutics
Gabor Veres, Applied Genetic Technologies
J. Fraser Wright, University of Pennsylvania School of Medicine
Yili Xu, Sangamo BioSciences
Olga Zelenaia, Children's Hospital of Philadelphia
Lorena Zentilin, International Center for Genetic Engineering and Biotechnology
Richard O. Snyder, University of Pennsylvania School of Medicine

UMMS Affiliation

Gene Therapy Center; Department of Pediatrics

Publication Date


Document Type



Biological Assay; DNA, 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


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.

DOI of Published Version



Hum Gene Ther. 2010 Oct;21(10):1273-85. Link to article on publisher's site

Journal/Book/Conference Title

Human gene therapy

Related Resources

Link to Article in PubMed

PubMed ID