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Published on: May 6, 2015
A three-plasmid system for construction of armed oncolytic adenovirus
Hong-Yan Liu1, Bing-Juan Han, Yu-Xu Zhong
1Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Abstract:
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.
Insights
A novel three-plasmid system simplifies the creation of armed oncolytic adenoviruses (Ads) for cancer gene therapy. This robust method enables the efficient construction of transcriptionally regulated oncolytic Ads carrying therapeutic genes.
Area of Science:
- Oncolytic virotherapy
- Adenovirus vector engineering
- Cancer gene therapy
Background:
- Oncolytic viruses are promising tools for cancer gene therapy, but their construction, particularly adenoviruses (Ads), faces challenges due to a lack of efficient methods.
- Developing robust and convenient systems for engineering oncolytic Ads is crucial for advancing their clinical application.
Purpose of the Study:
- To introduce and validate a novel three-plasmid system for the construction of armed oncolytic adenoviruses.
- To demonstrate the system's utility in generating transcriptionally regulated oncolytic Ads carrying therapeutic genes.
Main Methods:
- A three-plasmid system was developed, incorporating pShuttle-CMV, pAdEasy-1, and a new plasmid (pTE-ME1) containing the Ad5 E1 region with a modified promoter.
- Therapeutic genes and tissue-specific promoters (TSPs) were inserted into the shuttle plasmid and pTE-ME1, respectively. The modified E1 region was integrated into the shuttle plasmid.
- The final shuttle plasmid was recombined with pAdEasy-1 in E. coli to create the Ad plasmid, followed by rescue in packaging cells.
Main Results:
- The system successfully generated two oncolytic adenoviruses, Ad-GFP-TPE and Ad-GFP-D19K, using the green fluorescent protein (GFP) as a transgene and the telomerase reverse transcriptase promoter (TERTp) as a TSP.
- The generated oncolytic Ads demonstrated significant oncolytic and replicating abilities in TERT-positive tumor cells.
- The feasibility of constructing other transcriptionally regulated oncolytic Ads with therapeutic genes using this system was confirmed.
Conclusions:
- The developed three-plasmid system provides a practicable and efficient approach for constructing armed oncolytic adenoviruses.
- This system facilitates the routine generation of transcriptionally regulated oncolytic Ads, expanding the toolkit for cancer gene therapy.
- The findings support the potential of this method for developing novel oncolytic virotherapy strategies.
