Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods.
BACKGROUND
High-capacity adenoviruses (HC-Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large-scale production, and the absence of standardized methods for production and titration. In the present study, we compare the different methods found in the literature for characterizing HC-Ad production.


METHODS
Two productions of the HC-Ad carrying murine IL-12 gene were obtained. The viral titer and adenovirus-helper contamination as well as viral particle concentration of both productions were determined using different methods: (i) quantification of total viral particles by spectrophotometry and plaque assay to estimate first-generation (FG)-helper-Ad contamination; (ii) quantification of HC-Ad and FG-helper-Ad genomes by the quantitative polymerase chain reaction (qPCR) directly from viral stock; (iii) quantification of viral genomes after cell infection by the slot-blot hybridization assay and (iv) qPCR.


RESULTS
Dramatic differences with respect to viral titer were found depending on the method used. The first method overestimates HC-Ad titer and underestimates FG-helper-Ad contamination and no information on the infectivity of the HC-Ad is obtained. qPCR analysis of viral stock is more sensitive and accurate, but information about infectivity remains unknown and FG-helper-Ad contamination is overestimated. Quantification of HC-Ad and FG-helper-Ad infectious units by-slot blot DNA hybridization and qPCR assay are found to be equally sensitive and accurate.


CONCLUSIONS
The results of the present study demonstrate that a standardized method should be developed for HC-Ad characterization for future clinical applications of this vector. Quantification of HC-Ad production by qPCR is a fast, safe and reliable method for determining HC-Ad and FG-helper-Ad particles and infectious units.
Crettaz
Julien
J
Division of Hepatology and Gene Therapy, Center for Research in Applied Medicine, University of Navarra, Pamplona, Spain.
Olague
Cristina
C
Vales
Africa
A
Aurrekoetxea
Igor
I
Berraondo
Pedro
P
Otano
Itziar
I
Kochanek
Stephan
S
Prieto
Jesús
J
González-Aseguinolaza
Gloria
G
D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D004279:DNA, Viral / Q000032:analysis; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction / Q000592:standards*
D004279:DNA, Viral; D018664:Interleukin-12