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Updated: Mar 7, 2026
Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus
Published on: May 13, 2021
A Strategy for Cultivation of Retargeted Oncolytic Herpes Simplex Viruses in Non-cancer Cells
Valerio Leoni1, Valentina Gatta1, Costanza Casiraghi1
1Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy.
Abstract:
The oncolytic herpes simplex virus (HSV) that has been approved for clinical practice and those HSVs in clinical trials are attenuated viruses, often with the neurovirulence gene γ134.5 and additional genes deleted. One strategy to engineer nonattenuated oncolytic HSVs consists of retargeting the viral tropism to a cancer-specific receptor of choice, exemplified by HER2 (human epidermal growth factor receptor 2), which is present in breast, ovary, and other cancers, and in detargeting from the natural receptors. Because the HER2-retargeted HSVs strictly depend on this receptor for infection, the viruses employed in preclinical studies were cultivated in HER2-positive cancer cells. The production of clinical-grade viruses destined for humans should avoid the use of cancer cells. Here, we engineered the R-213 recombinant, by insertion of a 20-amino-acid (aa) short peptide (named GCN4) in the gH of R-LM113; this recombinant was retargeted to HER2 through insertion in gD of a single-chain antibody (scFv) to HER2. Next, we generated a Vero cell line expressing an artificial receptor (GCN4R) whose N terminus consists of an scFv to GCN4 and therefore is capable of interacting with GCN4 present in gH of R-213. R-213 replicated as well as R-LM113 in SK-OV-3 cells, implying that addition of the GCN4 peptide was not detrimental to gH. R-213 grew to relatively high titers in Vero-GCN4R cells, efficiently spread from cell to cell, and killed both Vero-GCN4R and SK-OV-3 cells, as expected for an oncolytic virus. Altogether, Vero-GCN4R cells represent an efficient system for cultivation of retargeted oncolytic HSVs in non-cancer cells.IMPORTANCE There is growing interest in viruses as oncolytic agents, which can be administered in combination with immunotherapeutic compounds, including immune checkpoint inhibitors. The oncolytic HSV approved for clinical practice and those in clinical trials are attenuated viruses. An alternative to attenuation is a cancer specificity achieved by tropism retargeting to selected cancer receptors. However, the retargeted oncolytic HSVs strictly depend on cancer receptors for infection. Here, we devised a strategy for in vitro cultivation of retargeted HSVs in non-cancer cells. The strategy envisions a double-retargeting approach: one retargeting is via gD to the cancer receptor, and the second retargeting is via gH to an artificial receptor expressed in Vero cells. The double-retargeted HSV uses alternatively the two receptors to infect cancer cells or producer cells. A universal non-cancer cell line for growth of clinical-grade retargeted HSVs represents a step forward in the translational phase.
Insights
Researchers developed a novel method to grow cancer-targeting oncolytic herpes simplex viruses (HSVs) in non-cancer cells. This breakthrough utilizes a double-retargeting strategy for efficient production of clinical-grade HSVs.
Area of Science:
- Virology
- Oncolytic Virotherapy
- Molecular Engineering
Background:
- Oncolytic herpes simplex viruses (HSVs) are promising cancer therapeutics, but current clinical candidates are attenuated.
- Retargeting HSVs to cancer-specific receptors like HER2 offers an alternative to attenuation but requires cancer cell cultivation.
- Clinical-grade virus production necessitates non-cancer cell systems to avoid contamination and ensure safety.
Purpose of the Study:
- To engineer a novel oncolytic HSV (R-213) with dual retargeting capabilities for cancer cells and a specific artificial receptor.
- To develop a non-cancer cell line (Vero-GCN4R) for efficient cultivation of the engineered oncolytic HSV.
- To demonstrate the feasibility of producing clinical-grade retargeted HSVs in a non-cancer cell system.
Main Methods:
- Engineered R-213 HSV by modifying glycoproteins gD (retargeting to HER2) and gH (incorporating GCN4 peptide).
- Created a Vero cell line expressing a GCN4-binding artificial receptor (GCN4R).
- Assessed R-213 replication, spread, and cell killing in both cancer (SK-OV-3) and engineered Vero-GCN4R cells.
Main Results:
- R-213 demonstrated efficient replication and cell killing in HER2-positive SK-OV-3 cancer cells.
- R-213 achieved high titers and efficient cell-to-cell spread in the engineered Vero-GCN4R cells.
- The GCN4 peptide insertion in gH did not impede viral replication compared to the parental virus.
Conclusions:
- The engineered R-213 HSV effectively utilizes dual retargeting for infection of cancer cells and cultivation in engineered non-cancer cells.
- Vero-GCN4R cells provide an efficient system for producing clinical-grade, retargeted oncolytic HSVs.
- This strategy represents a significant advancement for the translational development of oncolytic HSV therapies.
