Abstract
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. Copyright © 2008 John Wiley & Sons, Ltd.