Engineering a novel HSV-1 strain for oncolytic therapy of solid tumors

Abstract

Oncolytic herpes simplex virus (HSV)-1-derived viruses are being developed for cancer treatment. Here, we describe the isolation of a novel strain of HSV-1 and its engineering to safely harness it as an oncolytic therapeutic. This strain (UT1a) was isolated from a de-identified consented patient biorepository. CRISPR-Cas9-based recombination was utilized to insert bacterial artificial chromosome (BAC) genes into the viral UL39 and UL40 locus, resulting in the deletion of both large and small subunits of the viral ribonucleotide reductase (RR). Subsequent deletion of viral RL1 genes encoding the neurovirulence factor γ34.5 resulted in OncoDelta (OncoD), a virus deleted for UL39, UL40, and both copies of RL1. OncoD retained tumor-cell-specific cytotoxicity and replication; was safe and non-toxic in intracranial injections in naive mice up to doses of 5 × 10⁶, the maximal injectable dose for OncoD; and showed significant anti-tumor immune-activating potential in multiple tumor models. Transcriptome profiling of OncoD showed that it impaired DNA damage repair pathways and hence synergized with radiation to improve therapeutic response in vitro and in vivo.

Keywords: DNA damage; cancer therapy; herpes simplex virus; irradiation; oncolytic HSV-1; solid tumor.

Graphical abstract

Figure 1

Generation of triple-mutated oncolytic HSV-1 OncoD from UT1a strain (A) HSV-1 strain UT1a was isolated from a consented patient’s labial lesion and propagated in Vero cells. (B) Human tumor cells MDA-MB-468 and HCT116 and mouse DB7 cells were infected with UT1a and F-strain HSV-1 at the indicated MOIs for 72 h. (C) Schematic of mutations in UT1a to generate OncoD. (D) Immunofluorescence imaging of Vero cells transfected with UT-BAC with and without Cre-mediated recombination to remove the bacterial sequences, including GFP. Scale bar, 100 μm. (E and F) Analysis of OncoD replication in human cell lines GBM28 (E) and A549 (F) at the indicated MOIs. (G and H) Cytotoxicity analysis of OncoD against human tumor cells GBM12 (G) and HCT116 (H). ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 2

OncoD is safe at high doses in mice and improves survival in multiple in vivo mouse tumor models (A) Effect of OncoD on syngeneic A20 subcutaneous tumor growth in BALB/c mice. Once tumors reached the indicated tumor volume, OncoD or PBS was injected intratumorally at the indicated time points (green arrows). After treatment, tumor volumes were measured every other day. (B) Kaplan-Meier curve showing the proportion of mice reaching the established endpoint tumor volume over time. (C and D) Effect of OncoD on syngeneic MC38 (C) and LLC (D) subcutaneous tumor growth in C57BL6 mice. (E) Kaplan-Meier curve showing the effect of OncoD on the survival of NSG mice bearing intracranial human GBM12 tumors. OncoD was administered intratumorally at the indicated time points (green arrows). (F) Kaplan-Meier curve showing the effect of OncoD on the survival of C57BL6 mice bearing 005 mouse glioma tumors. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 3

OncoD induces DNA damage in infected tumor cells and synergizes with radiation (A–D) Heatmaps and significantly enriched gene set enrichment analysis (GSEA) pathways altered upon treatment with OncoD in the indicated cells. (E–G) Western blot (GBM12 and GBM28 cells, E) and immunofluorescence analysis (GBM12 cells, F) for γH2AX phosphorylation after treatment with OncoD and quantification of γH2AX foci (G). Scale bar, 10 μm. (H–J) Reduced colony formation in the indicated glioma cells after treatment with radiation (4 Gy) and OncoD. Scale bar, 200 μm. (K) Kaplan-Meier survival curve showing the effect of OncoD combination with IR in the mouse glioma 005 model. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.