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Updated: Aug 14, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region
Hidetaka A Ono1, Long P Le, Julia G Davydova
1Division of Human Gene Therapy, Department of Medicine, and the Gene Therapy Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Abstract:
To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.
Insights
Researchers developed a novel adenovirus vector that uses enhanced green fluorescent protein (EGFP) to track tumor-specific viral replication. This noninvasive imaging method allows real-time monitoring of oncolytic adenovirus activity in vivo.
Area of Science:
- Oncolytic virotherapy
- Molecular imaging
- Adenovirus engineering
Background:
- Current cancer treatments have limitations, including inefficacy and side effects.
- Conditionally replicative adenoviruses offer tumor-specific oncolysis.
- Lack of noninvasive methods hinders understanding of in vivo adenovirus replication.
Purpose of the Study:
- To develop a reporter system for noninvasive monitoring of adenovirus replication in vivo.
- To assess the correlation between reporter expression and viral replication.
Main Methods:
- Constructed adenovirus replication reporter vectors incorporating the enhanced green fluorescent protein (EGFP) gene in the E3 region.
- Maintained expression of the adenovirus death protein to preserve oncolytic function.
- Utilized fluorescence imaging and Western blot analysis to quantify viral replication and reporter expression.
Main Results:
- EGFP fluorescence was detected in a replication-dependent manner, correlating with viral DNA replication.
- In vivo fluorescence imaging successfully detected and monitored adenovirus replication noninvasively.
- Reporter gene expression accurately reflected viral DNA replication levels in tumors.
Conclusions:
- Adenovirus vectors expressing reporter genes provide a viable method for noninvasive monitoring of replication.
- This approach enables real-time assessment of oncolytic adenovirus activity in preclinical studies.
- The technology holds potential for clinical application with relevant imaging reporters.
