Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
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 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 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.
Lock
Martin
M
Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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
D001681:Biological Assay; D004279:DNA, Viral / Q000737:chemistry; D000229:Dependovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000302:isolation & purification; D016679:Genome, Viral; D006378:Helper Viruses; D016133:Polymerase Chain Reaction; D012015:Reference Standards; D014161:Transduction, Genetic; D014779:Virus Replication
D004279:DNA, Viral