A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes

• Published in: Cancer gene therapy Vol. 15; no. 3; pp. 173 - 182
• Main Authors: Hu, Z-B, Wu, C-T, Wang, H, Zhang, Q-W, Wang, L, Wang, R-L, Lu, Z-Z, Wang, L-S
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-03-2008; Nature Publishing Group
• Subjects: Adenoviridae - genetics; Adenovirus; Adenoviruses; Animals; Antitumor activity; B7-1 Antigen - genetics; Biomedical and Life Sciences; Biomedicine; Cancer; Care and treatment; CD80 antigen; Cell Line; Cell Line, Tumor; Cell Survival - genetics; Cell Survival - physiology; Colony-stimulating factor; E1A protein; E1B gene; Escherichia coli; Gene Expression; Gene Therapy; Genetic aspects; Genetic Therapy - methods; Genetic Vectors - genetics; Granulocyte-macrophage colony-stimulating factor; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Health aspects; HT29 Cells; Humans; Internal ribosome entry site; Male; Methods; Mice; Mice, Inbred BALB C; Mice, Nude; Multiplicity of infection; Oncogenic viruses; Oncolysis; Oncolytic Virotherapy - methods; Oncolytic Viruses - genetics; Oncolytic Viruses - physiology; original-article; Packaging; Plasmids; RNA-directed DNA polymerase; Telomerase; Telomerase reverse transcriptase; Transcription; Transgenes; Transgenes - genetics; Tumor cell lines; Tumor cells; Virus Replication - genetics; Virus Replication - physiology; Viruses; Xenograft Model Antitumor Assays; Xenografts; China; transgene; oncolytic; vector construction; adenovirus
• ISSN: 0929-1903; 1476-5500
• DOI: 10.1038/sj.cgt.7701105

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 × 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.