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Updated: Feb 2, 2026
Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus
Published on: May 13, 2021
A Novel Oncolytic Herpes Simplex Virus Design based on the Common Overexpression of microRNA-21 in Tumors
M Marzulli1, L Mazzacurati1, M Zhang1
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh.
Background:
Recognition sequences for microRNAs (miRs) that are down-regulated in tumor cells have recently been used to render lytic viruses tumor-specific. Since different tumor types down-regulate different miRs, this strategy requires virus customization to the target tumor. We have explored a feature that is shared by many tumor types, the up-regulation of miR-21, as a means to generate an oncolytic herpes simplex virus (HSV) that is applicable to a broad range of cancers.
Methods:
We assembled an expression construct for a dominant-negative (dn) form of the essential HSV replication factor UL9 and inserted tandem copies of the miR-21 recognition sequence (T21) in the 3' untranslated region. Bacterial Artificial Chromosome (BAC) recombineering was used to introduce the dnUL9 construct with or without T21 into the HSV genome. Virus was produced by transfection and replication was assessed in different tumor and control cell lines.
Results:
Virus production was conditional on the presence of the T21 sequence. The dnUL9-T21 virus replicated efficiently in tumor cell lines, less efficiently in cells that contained reduced miR-21 activity, and not at all in the absence of miR-21.
Conclusion:
miR-21-sensitive expression of a dominant-negative inhibitor of HSV replication allows preferential destruction of tumor cells in vitro. This observation provides a basis for further development of a widely applicable oncolytic HSV.
Insights
This study engineered an oncolytic herpes simplex virus (HSV) that targets cancer by utilizing the elevated miR-21 levels found in many tumors. The modified virus selectively replicates in cancer cells, offering a broadly applicable oncolytic therapy.
Area of Science:
- Oncolytic virotherapy
- Cancer gene therapy
- Molecular virology
Background:
- Tumor-specific viruses are engineered using microRNA (miR) recognition sequences.
- Current strategies require customization for different tumor types due to varying miR expression.
- miR-21 is upregulated in many cancer types, presenting an opportunity for broad-spectrum oncolytic virus development.
Purpose of the Study:
- To develop a broadly applicable oncolytic herpes simplex virus (HSV) targeting the common tumor feature of miR-21 upregulation.
- To engineer an HSV that selectively replicates in cancer cells based on miR-21 levels.
Main Methods:
- An expression construct for a dominant-negative (dn) UL9 HSV replication factor was created.
- Tandem copies of the miR-21 recognition sequence (T21) were inserted into the 3' untranslated region of the dnUL9 construct.
- Bacterial Artificial Chromosome (BAC) recombineering was used to introduce the construct into the HSV genome, followed by virus production and replication assessment.
Main Results:
- Virus production was dependent on the presence of the T21 sequence.
- The dnUL9-T21 virus demonstrated efficient replication in tumor cell lines.
- Replication was reduced in cells with lower miR-21 activity and absent in miR-21-deficient cells.
Conclusions:
- miR-21-sensitive expression of a dominant-negative HSV replication inhibitor enables preferential tumor cell destruction in vitro.
- This approach provides a foundation for developing a widely applicable oncolytic HSV for cancer therapy.
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