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

被引：108

作者：

Lock, Martin ^{[2
]}

McGorray, Susan ^{[3
]}

Auricchio, Alberto ^{[4
,5
]}

Ayuso, Eduard ^{[6
,7
]}

Beecham, E. Jeffrey ^{[8
]}

Blouin-Tavel, Veronique ^{[9
]}

Bosch, Fatima ^{[6
,7
]}

Bose, Mahuya ^{[10
]}

Byrne, Barry J. ^{[11
,12
]}

Caton, Tina ^{[10
]}

Chiorini, John A. ^{[13
]}

Chtarto, Abdelwahed ^{[14
,15
]}

Clark, K. Reed ^{[16
,17
]}

Conlon, Thomas ^{[12
]}

Darmon, Christophe ^{[9
]}

Doria, Monica ^{[5
]}

Douar, Anne ^{[18
]}

Flotte, Terence R. ^{[19
,20
]}

Francis, Joyce D. ^{[10
]}

Francois, Achille ^{[9
]}

Giacca, Mauro ^{[21
]}

Korn, Michael T. ^{[2
]}

Korytov, Irina ^{[12
]}

Leon, Xavier ^{[6
,7
]}

Leuchs, Barbara ^{[22
]}

Lux, Gabriele ^{[23
]}

Melas, Catherine ^{[14
,15
]}

Mizukami, Hiroaki ^{[24
]}

Moullier, Philippe ^{[9
]}

Mueller, Marcus ^{[22
]}

Ozawa, Keiya ^{[24
]}

Philipsberg, Tina ^{[12
]}

Poulard, Karine ^{[18
]}

Raupp, Christina ^{[22
]}

Riviere, Christel ^{[18
]}

Roosendaal, Sigrid D. ^{[25
]}

Samulski, R. Jude ^{[8
]}

Soltys, Steven M. ^{[8
]}

Surosky, Richard ^{[26
]}

Tenenbau, Liliane ^{[14
,15
]}

Thomas, Darby L. ^{[27
]}

van Montfort, Bart ^{[25
]}

Veres, Gabor ^{[27
]}

Wright, J. Fraser ^{[28
,29
]}

Xu, Yili ^{[26
]}

Zelenaia, Olga ^{[28
]}

Zentilin, Lorena ^{[21
]}

Snyder, Richard O. ^{[1
,10
,11
]}

机构：

[1] Univ Florida, Coll Med, Dept Mol Genet & Microbiol, Gainesville, FL 32610 USA

[2] Univ Penn, Dept Pathol & Lab Med, Gene Therapy Program, Philadelphia, PA 19104 USA

[3] Univ Florida, Coll Med, Dept Epidemiol & Hlth Policy Res, Gainesville, FL 32610 USA

[4] Univ Naples Federico II, Dept Pediat, I-80131 Naples, Italy

[5] Telethon Inst Genet & Med TIGEM, I-80131 Naples, Italy

[6] Univ Autonoma Barcelona, Sch Vet Med, Ctr Anim Biotechnol & Gene Therapy, Bellaterra 08193, Spain

[7] Univ Autonoma Barcelona, Sch Vet Med, Dept Biochem & Mol Biol, Bellaterra 08193, Spain

[8] Univ N Carolina, Gene Therapy Ctr, Chapel Hill, NC 27599 USA

[9] CHU Nantes, INSERM, UMR649, F-44007 Nantes, France

[10] Univ Florida, Ctr Excellence Regenerat Hlth Biotechnol, Gainesville, FL 32611 USA

[11] Univ Florida, Dept Pediat, Div Cellular & Mol Therapy, Gainesville, FL 32610 USA

[12] Univ Florida, Powell Gene Therapy Ctr Vector Core, Gainesville, FL 32611 USA

[13] Natl Inst Dent & Craniofacial Res, Mol Physiol & Therapeut Branch, NIH, Bethesda, MD 20892 USA

[14] Free Univ Brussels, Lab Expt Neurosurg, B-1070 Brussels, Belgium

[15] Free Univ Brussels, Multidisciplinary Res Inst, B-1070 Brussels, Belgium

[16] Nationwide Childrens Hosp, Res Inst, Ctr Gene Therapy, Columbus, OH 43205 USA

[17] Ohio State Univ, Dept Pediat, Columbus, OH 43210 USA

[18] Genethon, F-91000 Evry, France

[19] Univ Massachusetts, Sch Med, Gene Therapy Ctr, Worcester, MA 01655 USA

[20] Univ Massachusetts, Dept Pediat, Worcester, MA 01655 USA

[21] ICGEB, Mol Med Lab, I-34149 Trieste, Italy

[22] German Canc Res Ctr, Infect & Canc Program, D-69120 Heidelberg, Germany

[23] Pharmazeut Fabrik Dr Reckeweg, D-64625 Bensheim, Germany

[24] Jichi Med Univ, Ctr Mol Med, Div Genet Therapeut, Shimotsuke 3290498, Japan

[25] Amsterdam Mol Therapeut, NL-1100 Amsterdam, Netherlands

[26] Sangamo BioSci, Richmond, CA 94804 USA

[27] Appl Genet Technol, Alachua, FL 32615 USA

[28] Childrens Hosp Philadelphia, Ctr Cellular & Mol Therapeut, Philadelphia, PA 19104 USA

[29] Univ Penn, Sch Med, Dept Pathol & Lab Med, Philadelphia, PA 19104 USA

来源：

HUMAN GENE THERAPY | 2010年 / 21卷 / 10期

关键词：

GENE-THERAPY; VIRAL VECTORS; PURIFICATION; SEROTYPE-2; CAPSIDS; TRIAL;

D O I：

10.1089/hum.2009.223

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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(11) particles/ml; 95% confidence interval [CI], 7.89 x 10(11) to 1.05 x 10(12) particles/ml), vector genomes ({X}, 3.28 x 10(10) vector genomes/ml; 95% CI, 2.70 x 10(10) to 4.75 x 10(10) vector genomes/ml), transducing units ({X}, 5.09 x 10(8) transducing units/ml; 95% CI, 2.00 x 10(8) to 9.60 x 10(8) transducing units/ml), and infectious units ({X}, 4.37 x 10(9) TCID50 IU/ml; 95% CI, 2.06 x 10(9) to 9.26 x 10(9) 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.

引用

页码：1273 / 1285

页数：13

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