title: A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes.
abstract: Oncolytic adenoviruses, also called conditionally replicating adenoviruses (CRADs), have been widely applied in cancer gene therapy. However, the construction of CRADs is still time-consuming. In this study, we attempted to establish a simplified method of generating CRADs based on AdEasy system. A novel plasmid pTE-TPE-GM was constructed, containing sequentially positioned promoter of telomerase reverse transcriptase (TERTp), coding sequence of E1A gene, promoter of E1B gene, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene, internal ribosome entry site sequence and coding sequence of E1B55K gene. The CRAD-generating system reported here include three plasmids: pTE-TPE-GM, pShuttle-CMV and AdEasy-1, one Escherichia coli strain BJ5183, and the packaging cell line 293. Using this system, an oncolytic adenovirus carrying B7-1 (CD80) and GM-CSF genes was successfully constructed and designated as Ad-CD80-TPE-GM. The expression of GM-CSF increased more than 9000 times in tumor cell lines infected by Ad-CD80-TPE-GM at a multiplicity of infection (MOI) of 5, compared with the cells infected by replication-defective control virus. Similarly, the expression of CD80 also increased 9-140 times. Ad-CD80-TPE-GM selectively replicates in TERT-positive tumor cells, and the progeny viruses can reach up to 375 infection units (IU) per cell. In vitro study showed that the Ad-CD80-TPE-GM induced an obvious oncolytic effect at MOI of 0.1, and killed about 80% TERT-positive tumor cells within 7 days at an MOI of 1. The antitumor effect of this vector was also investigated in Hep2 xenograft model of nude mice, and the tumor inhibition rate reached 74% at day 30 after the administration with a total dose of 1 x 10(9) IU Ad-CD80-TPE-GM. Intratumoral injection of Ad-CD80-TPE-GM slightly induced neutralizing antibody against the oncolytic adenovirus in nude mice, which might contribute to the virus clearance in vivo. In conclusion, we successfully constructed an oncolytic CRAD carrying GM-CSF and CD80 gene. More importantly, this system can be modified to generate novel transcriptionally regulated CRADs with different tissue-specific promoters or transgenes.
authors.affiliation: Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, PR China.
authors.forename: Z-B
authors.initials: ZB
authors.lastname: Hu
authors.forename: C-T
authors.initials: CT
authors.lastname: Wu
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: Q-W
authors.initials: QW
authors.lastname: Zhang
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: R-L
authors.initials: RL
authors.lastname: Wang
authors.forename: Z-Z
authors.initials: ZZ
authors.lastname: Lu
authors.forename: L-S
authors.initials: LS
authors.lastname: Wang
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D019073:HT29 Cells; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D050130:Oncolytic Virotherapy / Q000379:methods; D050504:Oncolytic Viruses / Q000235:genetics* / Q000502:physiology; D019076:Transgenes / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology; D023041:Xenograft Model Antitumor Assays
chemical_list: D018122:B7-1 Antigen; D016178:Granulocyte-Macrophage Colony-Stimulating Factor