A three-plasmid system for construction of armed oncolytic adenovirus.
There is growing interest in the use of oncolytic virus as a tool in cancer gene therapy. However, construction of oncolytic adenovirus (Ad) is not an easy task due to lack of convenient, robust methods. A three-plasmid system was introduced for construction of armed oncolytic Ad. Besides the pShuttle-CMV and pAdEasy-1, a third plasmid (pTE-ME1), harboring the E1 region of Ad5, was generated and included in this system. In pTE-ME1, the promoter of E1A was deleted and replaced with a multiple-cloning site (MCS). A therapeutic gene and tissue-specific promoter (TSP) could be inserted routinely into the MCS of pShuttle-CMV and pTE-ME1, respectively. The modified E1 region could then be excised from pTE-ME1 and integrated into the therapeutic gene-containing pShuttle-CMV to form the final shuttle plasmid. This shuttle plasmid was recombined with pAdEasy-1 in Escherichia coli strain BJ5183 to generate Ad plasmid. Finally, the oncolytic Ad could be rescued in Ad plasmid-transfected packaging cells. The GFP gene and the promoter of telomerase reverse transcriptase (TERTp) were chosen as the transgene and TSP, respectively, to test this system. Two oncolytic Ads, Ad-GFP-TPE and Ad-GFP-D19K, were generated successfully. Their oncolytic and replicating abilities were investigated in TERT-positive tumor cells. The results suggest that the three-plasmid system was practicable and could be used to construct other transcriptionally regulated oncolytic Ads carrying a therapeutic gene.
Liu
Hong-Yan
HY
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Han
Bing-Juan
BJ
Zhong
Yu-Xu
YX
Lu
Zhuo-Zhuang
ZZ
D017501:Adenovirus E1B Proteins / Q000235:genetics; D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D045744:Cell Line, Tumor; D005347:Fibroblasts / Q000821:virology; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000821:virology; D050504:Oncolytic Viruses / Q000235:genetics / Q000502:physiology; D009928:Organ Specificity; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D019098:Telomerase / Q000235:genetics; D014162:Transfection; D019076:Transgenes; D014779:Virus Replication
D017501:Adenovirus E1B Proteins; D049452:Green Fluorescent Proteins; C509186:TERT protein, human; D019098:Telomerase