Characterization of a recombinant adeno-associated virus type 2 reference standard material

Martin Lock; Susan McGorray; Alberto Auricchio; Eduard Ayuso; E. Jeffrey Beecham; Véronique Blouin-Tavel; Fatima Bosch; Mahuya Bose; Barry J. Byrne; Tina Caton; John A. Chiorini; Abdelwahed Chtarto; K. Reed Clark; Thomas Conlon; Christophe Darmon; Monica Doria; Anne Douar; Terence R. Flotte; Joyce D. Francis; Achille Francois; Mauro Giacca; Michael T. Korn; Irina Korytov; Xavier Leon; Barbara Leuchs; Gabriele Lux; Catherine Melas; Hiroaki Mizukami; Philippe Moullier; Marcus Müller; Keiya Ozawa; Tina Philipsberg; Karine Poulard; Christina Raupp; Christel Rivière; Sigrid D. Roosendaal; R. Jude Samulski; Steven M. Soltys; Richard Surosky; Liliane Tenenbaum; Darby L. Thomas; Bart Van Montfort; Gabor Veres; J. Fraser Wright; Yili Xu; Olga Zelenaia; Lorena Zentilin; Richard O. Snyder

doi:https://doi.org/10.1089/hum.2009.223

Characterization of a recombinant adeno-associated virus type 2 reference standard material

Martin Lock, Susan McGorray, Alberto Auricchio, Eduard Ayuso, E. Jeffrey Beecham, Véronique Blouin-Tavel, Fatima Bosch, Mahuya Bose, Barry J. Byrne, Tina Caton, John A. Chiorini, Abdelwahed Chtarto, K. Reed Clark, Thomas Conlon, Christophe Darmon, Monica Doria, Anne Douar, Terence R. Flotte, Joyce D. Francis, Achille FrancoisMauro Giacca, Michael T. Korn, Irina Korytov, Xavier Leon, Barbara Leuchs, Gabriele Lux, Catherine Melas, Hiroaki Mizukami, Philippe Moullier, Marcus Müller, Keiya Ozawa, Tina Philipsberg, Karine Poulard, Christina Raupp, Christel Rivière, Sigrid D. Roosendaal, R. Jude Samulski, Steven M. Soltys, Richard Surosky, Liliane Tenenbaum, Darby L. Thomas, Bart Van Montfort, Gabor Veres, J. Fraser Wright, Yili Xu, Olga Zelenaia, Lorena Zentilin, Richard O. Snyder

Research output: Contribution to journal › Article › Research › peer-review

82 Citations (Scopus)

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}, 9.18×1011 particles/ml; 95% confidence interval [CI], 7.89×1011 to 1.05×1012 particles/ml), vector genomes ({X}, 3.28×1010 vector genomes/ml; 95% CI, 2.70×1010 to 4.75×1010 vector genomes/ml), transducing units ({X}, 5.09×108 transducing units/ml; 95% CI, 2.00×108 to 9.60×108 transducing units/ml), and infectious units ({X}, 4.37×109 TCID50 IU/ml; 95% CI, 2.06×109 to 9.26×109 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. © 2010 Mary Ann Liebert, Inc.

Original language	English
Pages (from-to)	1273-1285
Journal	Human Gene Therapy
Volume	21
DOIs	https://doi.org/10.1089/hum.2009.223
Publication status	Published - 1 Oct 2010

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Lock, M., McGorray, S., Auricchio, A., Ayuso, E., Beecham, E. J., Blouin-Tavel, V., Bosch, F., Bose, M., Byrne, B. J., Caton, T., Chiorini, J. A., Chtarto, A., Clark, K. R., Conlon, T., Darmon, C., Doria, M., Douar, A., Flotte, T. R., Francis, J. D., ... Snyder, R. O. (2010). Characterization of a recombinant adeno-associated virus type 2 reference standard material. Human Gene Therapy, 21, 1273-1285. https://doi.org/10.1089/hum.2009.223

Lock, Martin ; McGorray, Susan ; Auricchio, Alberto ; Ayuso, Eduard ; Beecham, E. Jeffrey ; Blouin-Tavel, Véronique ; Bosch, Fatima ; Bose, Mahuya ; Byrne, 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 ; Müller, Marcus ; Ozawa, Keiya ; Philipsberg, Tina ; Poulard, Karine ; Raupp, Christina ; Rivière, Christel ; Roosendaal, Sigrid D. ; Samulski, R. 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. / Characterization of a recombinant adeno-associated virus type 2 reference standard material. In: Human Gene Therapy. 2010 ; Vol. 21. pp. 1273-1285.

@article{99d9371069f5407694b3c972cc868e3d,

title = "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}, 9.18×1011 particles/ml; 95% confidence interval [CI], 7.89×1011 to 1.05×1012 particles/ml), vector genomes ({X}, 3.28×1010 vector genomes/ml; 95% CI, 2.70×1010 to 4.75×1010 vector genomes/ml), transducing units ({X}, 5.09×108 transducing units/ml; 95% CI, 2.00×108 to 9.60×108 transducing units/ml), and infectious units ({X}, 4.37×109 TCID50 IU/ml; 95% CI, 2.06×109 to 9.26×109 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. {\textcopyright} 2010 Mary Ann Liebert, Inc.",

author = "Martin Lock and Susan McGorray and Alberto Auricchio and Eduard Ayuso and Beecham, {E. Jeffrey} and V{\'e}ronique Blouin-Tavel and Fatima Bosch and Mahuya Bose and Byrne, {Barry J.} and Tina Caton and Chiorini, {John A.} and Abdelwahed Chtarto and Clark, {K. Reed} and Thomas Conlon and Christophe Darmon and Monica Doria and Anne Douar and Flotte, {Terence R.} and Francis, {Joyce D.} and Achille Francois and Mauro Giacca and Korn, {Michael T.} and Irina Korytov and Xavier Leon and Barbara Leuchs and Gabriele Lux and Catherine Melas and Hiroaki Mizukami and Philippe Moullier and Marcus M{\"u}ller and Keiya Ozawa and Tina Philipsberg and Karine Poulard and Christina Raupp and Christel Rivi{\`e}re and Roosendaal, {Sigrid D.} and Samulski, {R. Jude} and Soltys, {Steven M.} and Richard Surosky and Liliane Tenenbaum and Thomas, {Darby L.} and {Van Montfort}, Bart and Gabor Veres and Wright, {J. Fraser} and Yili Xu and Olga Zelenaia and Lorena Zentilin and Snyder, {Richard O.}",

year = "2010",

month = oct,

day = "1",

doi = "https://doi.org/10.1089/hum.2009.223",

language = "English",

volume = "21",

pages = "1273--1285",

}

Lock, M, McGorray, S, Auricchio, A, Ayuso, E, Beecham, EJ, Blouin-Tavel, V, Bosch, F, Bose, M, Byrne, BJ, Caton, T, Chiorini, JA, Chtarto, A, Clark, KR, Conlon, T, Darmon, C, Doria, M, Douar, A, Flotte, TR, Francis, JD, Francois, A, Giacca, M, Korn, MT, Korytov, I, Leon, X, Leuchs, B, Lux, G, Melas, C, Mizukami, H, Moullier, P, Müller, M, Ozawa, K, Philipsberg, T, Poulard, K, Raupp, C, Rivière, C, Roosendaal, SD, Samulski, RJ, Soltys, SM, Surosky, R, Tenenbaum, L, Thomas, DL, Van Montfort, B, Veres, G, Wright, JF, Xu, Y, Zelenaia, O, Zentilin, L & Snyder, RO 2010, 'Characterization of a recombinant adeno-associated virus type 2 reference standard material', Human Gene Therapy, vol. 21, pp. 1273-1285. https://doi.org/10.1089/hum.2009.223

Characterization of a recombinant adeno-associated virus type 2 reference standard material. / Lock, Martin; McGorray, Susan; Auricchio, Alberto; Ayuso, Eduard; Beecham, E. Jeffrey; Blouin-Tavel, Véronique; Bosch, Fatima; Bose, Mahuya; Byrne, 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; Müller, Marcus; Ozawa, Keiya; Philipsberg, Tina; Poulard, Karine; Raupp, Christina; Rivière, Christel; Roosendaal, Sigrid D.; Samulski, R. 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.

In: Human Gene Therapy, Vol. 21, 01.10.2010, p. 1273-1285.

Research output: Contribution to journal › Article › Research › peer-review

TY - JOUR

T1 - Characterization of a recombinant adeno-associated virus type 2 reference standard material

AU - Lock, Martin

AU - McGorray, Susan

AU - Auricchio, Alberto

AU - Ayuso, Eduard

AU - Beecham, E. Jeffrey

AU - Blouin-Tavel, Véronique

AU - Bosch, Fatima

AU - Bose, Mahuya

AU - Byrne, Barry J.

AU - Caton, Tina

AU - Chiorini, John A.

AU - Chtarto, Abdelwahed

AU - Clark, K. Reed

AU - Conlon, Thomas

AU - Darmon, Christophe

AU - Doria, Monica

AU - Douar, Anne

AU - Flotte, Terence R.

AU - Francis, Joyce D.

AU - Francois, Achille

AU - Giacca, Mauro

AU - Korn, Michael T.

AU - Korytov, Irina

AU - Leon, Xavier

AU - Leuchs, Barbara

AU - Lux, Gabriele

AU - Melas, Catherine

AU - Mizukami, Hiroaki

AU - Moullier, Philippe

AU - Müller, Marcus

AU - Ozawa, Keiya

AU - Philipsberg, Tina

AU - Poulard, Karine

AU - Raupp, Christina

AU - Rivière, Christel

AU - Roosendaal, Sigrid D.

AU - Samulski, R. Jude

AU - Soltys, Steven M.

AU - Surosky, Richard

AU - Tenenbaum, Liliane

AU - Thomas, Darby L.

AU - Van Montfort, Bart

AU - Veres, Gabor

AU - Wright, J. Fraser

AU - Xu, Yili

AU - Zelenaia, Olga

AU - Zentilin, Lorena

AU - Snyder, Richard O.

PY - 2010/10/1

Y1 - 2010/10/1

N2 - 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}, 9.18×1011 particles/ml; 95% confidence interval [CI], 7.89×1011 to 1.05×1012 particles/ml), vector genomes ({X}, 3.28×1010 vector genomes/ml; 95% CI, 2.70×1010 to 4.75×1010 vector genomes/ml), transducing units ({X}, 5.09×108 transducing units/ml; 95% CI, 2.00×108 to 9.60×108 transducing units/ml), and infectious units ({X}, 4.37×109 TCID50 IU/ml; 95% CI, 2.06×109 to 9.26×109 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. © 2010 Mary Ann Liebert, Inc.

AB - 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}, 9.18×1011 particles/ml; 95% confidence interval [CI], 7.89×1011 to 1.05×1012 particles/ml), vector genomes ({X}, 3.28×1010 vector genomes/ml; 95% CI, 2.70×1010 to 4.75×1010 vector genomes/ml), transducing units ({X}, 5.09×108 transducing units/ml; 95% CI, 2.00×108 to 9.60×108 transducing units/ml), and infectious units ({X}, 4.37×109 TCID50 IU/ml; 95% CI, 2.06×109 to 9.26×109 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. © 2010 Mary Ann Liebert, Inc.

U2 - https://doi.org/10.1089/hum.2009.223

DO - https://doi.org/10.1089/hum.2009.223

M3 - Article

VL - 21

SP - 1273

EP - 1285

ER -