title: Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods.
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.
authors.affiliation: Division of Hepatology and Gene Therapy, Center for Research in Applied Medicine, University of Navarra, Pamplona, Spain.
authors.forename: Julien
authors.initials: J
authors.lastname: Crettaz
authors.forename: Cristina
authors.initials: C
authors.lastname: Olague
authors.forename: Africa
authors.initials: A
authors.lastname: Vales
authors.forename: Igor
authors.initials: I
authors.lastname: Aurrekoetxea
authors.forename: Pedro
authors.initials: P
authors.lastname: Berraondo
authors.forename: Itziar
authors.initials: I
authors.lastname: Otano
authors.forename: Stephan
authors.initials: S
authors.lastname: Kochanek
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Gloria
authors.initials: G
authors.lastname: González-Aseguinolaza
mesh_terms: 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*
chemical_list: D004279:DNA, Viral; D018664:Interleukin-12