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.