Attenuated Replication-Competent Herpes Simplex Virus Expressing an ECM-Modifying Transgene Hyaluronan Synthase 2 of Naked Mole Rat in Oncolytic Gene Therapy

Abstract

Herpes simplex virus (HSV) has proven successful in treating human cancer. Since the approval of talimogene laherparepvec (T-VEC) in 2015, HSV has been thoroughly researched to discover novel mechanisms to combat cancer and treat other diseases. Another HSV-based drug, beremagene geperpavec (B-VEC), received approval in 2023 to treat the rare genetic disease dystrophic epidermolysis bullosa, and was also the first clinically approved HSV vector carrying an extracellular matrix (ECM)-modifying transgene. The ECM is a network of macromolecules surrounding cells, which provides support and regulates cell growth and differentiation, the disruption of which is common in cancer. The naked mole rat (NMR) has a thick ECM and a unique mutation in the hyaluronan synthase 2 (HAS2) gene, which has been linked to the high cancer resistance of the species. To study the effect of this mutation in human cancer, we have developed an attenuated, replication-competent HSV vector expressing the NMR-HAS2 gene. The viral replication, transgene expression and cytotoxic effect of the novel vector was studied in glioma cells. Our results show that an attenuated, replication-competent HSV vector expressing a foreign ECM-modifying transgene, namely HAS2, provides an effective tool to study and combat cancer in humans.

Keywords: cancer; extracellular matrix; herpes simplex; hyaluronan synthase; naked mole rat; oncolytic virus.

Figure 1

Development pathway of γ₁34.5- and gC-negative HSV vectors H0951 and H1551. The light gray arrows represent HSV genes, the dark gray arrows represent the transgenes and the crosses represent gene deletions.

Figure 2

The replication patterns of H1551 (NMR-HAS2) and H0951 (GFP) in U373MG (a) and Vero (b) cells, based on extracellularly released progeny virus from 1 pfu/cell infection on a logarithmic scale. The closed circles and triangles in the graph represent the titer of the released virus. The capped bars represent the standard error of the mean (SEM) (n = 3). Time point of 0 hpi in the graph represents the titer of the medium used to infect the cells.

Figure 3

mRNA expression in glioma cell line U373MG infected with H1551 (NMR-HAS2) and H0951 (GFP). The expression patterns of NMR-HAS2 (a), VP16 (b) and PKR (c), normalized to housekeeping gene GAPDH (d), are shown on a logarithmic scale. The columns represent the copy numbers of the mRNA, and the capped bars represent the SEM (n = 3).

Figure 4

Cytotoxicity in glioma cell line U373MG. The cells were infected with 1 pfu/cell of H1551 (NMR-HAS2), H0951 (GFP) and LoxLuc gC- (no transgene in the γ₁34.5 locus) and measured for cell viability 24 hpi and 48 hpi, utilizing a luminescent assay. The viability was determined by comparing the signal derived from the infected cells to that derived from the non-infected cells. The columns represent the percentage of viability of the cells, and the capped bars represent the SEM (n = 3). H1551 (NMR-HAS2) showed higher cytotoxicity compared to that of the controls at both time points, and at the time point 48 hpi, the difference was statistically significant (p = 0.029).

Figure 5

Cytotoxicity in glioma cell line U373MG at 48 hpi. The cells were infected with 0.001–1 pfu/cell of H1551 (NMR-HAS2), H0951 (GFP) and LoxLuc gC- (no transgene in the γ₁34.5 locus) and measured for cell viability at 48 hpi, utilizing a luminescent assay. The viability was determined by comparing the signal derived from the infected cells to that from the non-infected cells. The closed circles represent the percentage of viability of the cells, and the capped bars represent the SEM (n = 3).

Figure 6

Cytotoxicity in glioma cell line U373MG. The cells were infected with 2 pfu/cell of H1551 (NMR-HAS2), in parallel with a study on the oncolytic potential of wild-type HSV strains, and measured for cell viability at 96 hpi, utilizing a luminescent assay. The viability was determined by comparing the signal derived from the infected cells to that derived from the non-infected cells. The columns and the closed diamonds represent the percentage of viability of the cells, and the capped bars represent the SEM (n = 4). The asterisks represent the data that was published earlier by Kalke et al. [11], the scatter plot on the left represents the cytotoxicity of the studied wild-type HSV strains and the column in the center represents HSV-1 17+, which is the γ₁34.5-positive parental strain from which H1551 is derived. The cytotoxic effect of H1551 was higher compared to each of the published wild-type HSV strains, and the difference was statistically significant (p = 0.013).