title: Establishment of biological and pharmacokinetic assays of telomerase-specific replication-selective adenovirus.
abstract: The use of replication-selective tumor-specific viruses represents a novel approach for the treatment of neoplastic disease. We constructed an attenuated adenovirus, telomerase-specific replication-selective adenovirus (TRAD), in which the human telomerase reverse transcriptase promoter element drives the expression of the E1A and E1B genes linked with an internal ribosome entry site (IRES). Forty-eight hours after TRAD infection at a multiplicity of infection of 1.0, the cell viability of H1299 human lung cancer cells was consistently less than 50% and therefore this procedure could be used as a potency assay to assess the biological activity of TRAD. We also established a quantitative real-time polymerase chain reaction (PCR) analysis with consensus primers for either the adenovirus E1A or IRES sequence. The linear ranges of quantitation with E1A and IRES primers were 10(3)-10(8) and 10(2)-10(8) plaque-forming units/mL in the plasma, respectively. The PCR analysis demonstrated that the levels of E1A in normal tissues were more than 10(3) lower than in the tumors of A549 human lung tumor xenografts in nu/nmicro mice at 28 days after intratumoral injection. Our results suggest that the cell-killing assay against H1299 cells and real-time PCR can be used to assess the biological activity and biodistribution of TRAD in clinical trials.
authors.affiliation: Oncolys BioPharma, 3-16-33 Roppongi, Minato-ku, Tokyo 106-0031, Japan.
authors.forename: Yuuri
authors.initials: Y
authors.lastname: Hashimoto
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Shirakiya
authors.forename: Futoshi
authors.initials: F
authors.lastname: Uno
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Kagawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Kawamura
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Nagai
authors.forename: Noriaki
authors.initials: N
authors.lastname: Tanaka
authors.forename: Horomi
authors.initials: H
authors.lastname: Kumon
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Urata
authors.forename: Toshiyoshi
authors.initials: T
authors.lastname: Fujiwara
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D004261:DNA Replication; D004279:DNA, Viral / Q000493:pharmacokinetics; D005260:Female; D016679:Genome, Viral; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D023421:Models, Animal; D050130:Oncolytic Virotherapy / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019098:Telomerase / Q000235:genetics*
chemical_list: D004279:DNA, Viral; D019098:Telomerase