Oncolytic Herpes Simplex Research Articles

Development and Preliminary Application of a Droplet Digital PCR Assay for Quantifying the Oncolytic Herpes Simplex Virus Type 1 in the Clinical-Grade Production.

Oncolytic herpes simplex virus (oHSV) is a type of virus that selectively targets and kills cancer cells, leaving normal cells unharmed. Accurate viral titer is of great importance for the production and application of oHSV products. Droplet digital PCR (ddPCR) is known for having good reproducibility, not requiring a standard curve, not being affected by inhibitors, and being precise even in the detection of low copies. In the present study, we developed a droplet digital PCR assay for the quantification of HSV-1 and applied it in the oHSV production. The established ddPCR showed good specificity, linearity, a low limit of quantification, great reproducibility, and accuracy. The quantification result was well-associated with that of plaque assay and CCID50. Amplification of the purified virus without DNA extraction by ddPCR presented similar results to that from the extracted DNA, confirming the good resistance against PCR inhibitors. With the ddPCR, viral titer could be monitored in real time during the production of oHSV; the optimal harvest time was determined for the best virus yield in each batch. The ddPCR can be used as a useful tool for the quantification of oHSV and greatly facilitate the manufacturing process of oHSV products.

Systemic delivery of glycosylated-PEG-masked oncolytic virus enhances targeting of antitumor immuno-virotherapy and modulates T and NK cell infiltration.

Rationale: Immuno-virotherapy has emerged as a promising approach for cancer treatment, as it directly and cytotoxically eliminates tumors with systemic immune stimulation. However, the clinical efficacy of this approach remains limited by inappropriate delivery routes, robust antiviral responses, and the tumor immunosuppressive microenvironment. Methods: To address these challenges, we propose a surface engineering strategy that masks oncolytic herpes simplex virus (oHSV) with a galactose-polyethylene-glycol (PEG) polymer chain to minimize host antiviral responses and selectively targets tumors by limiting exposure to circulation upon systemic administration. We evaluated the antitumor efficacy of glycosylated-PEG-oHSV by examining tumor growth in animal models and analyzing tumor-infiltrating CD8+T cells and NK cells in the tumor microenvironment (TME). To assess the neutralizing antibody levels after systemic administration of glycosylated-PEG-oHSV, we utilized a mouse model and measured oHSV-specific IgG. Results: We demonstrate that the glycosylated-PEG modified oHSV does not affect the replication of oHSV yet exhibits high specificity to the asialoglycoprotein receptor (ASGPR) overexpressed in hepatocellular carcinoma cells. This results in selectively targeting cancer cells and deep penetration into tumors while avoiding spreading into the brain. Our approach also effectively reduces oHSV-specific neutralizing antibody levels to mitigate host antiviral immune response. Notably, our glycosylated-PEG-oHSV alleviates the immunosuppressive microenvironment within tumors by reducing regulatory T cells, augmenting the infiltration of activated CD8+T cells and NK cells with increasing release of anti-tumor cytokines, to impede tumor progression. Conclusion: Our findings offer a widely applicable and universal strategy to enhance cancer immuno-virotherapy through systemic administration of non-genetically engineered oncolytic viruses. This approach has the potential to overcome the limitations of current immune-virotherapy strategies and may improve clinical outcomes for cancer patients.

European Pharmacopoeia's Approach to Cell and Gene Therapy: Focus on How Gene Therapy Texts Are Evolving.

The European Pharmacopoeia (Ph. Eur.) constitutes single recognised common standard for the quality control of medicines in Europe, which is also applied in many countries worldwide. In 2000, the European Pharmacopoeia Commission (EPC), the decision-making body of the Ph. Eur., set out to elaborate a text on gene therapy products. This resulted in the publication of the widely used and much appreciated general chapter Gene transfer medicinal products for human use (5.14) in 2006, at a time when no gene therapy medicinal products were yet approved on the European market. As a general chapter, it is not legally binding, but it reflects the consensus of the European authorities. Now, more than two decades after the first initiative in the gene therapy field and with several gene therapy medicinal products launched in Europe, the EPC proposes a modernised approach and the creation of a general monograph, i.e. a legally binding standard. In addition to general requirements for gene therapy medicinal products, it is also envisaged that this general monograph will outline specific criteria for the classes of products already on the market, i.e. genetically modified human autologous cells, adeno-associated virus vectors for human use and recombinant oncolytic herpes simplex viruses for human use. It is also proposed to create a new general chapter, Additional information on gene therapy medicinal products for human use (5.34), comprising the remaining sections of Chap. 5.14, as well as a newly elaborated section on genetically modified bacterial cells.

Histone Deacetylase Inhibitor Panobinostat Benefits the Therapeutic Efficacy of Oncolytic Herpes Simplex Virus Combined with PD-1/PD-L1 Blocking in Glioma and Squamous Cell Carcinoma Models.

Combination therapy has been widely explored for oncolytic virus (OV), as it can be met with tumor resistance. The HDAC inhibitor (HDACi) panobinostat is a potent pan-deacetylase inhibitor which blocks multiple cancer-related pathways and reverses epigenetic events in cancer progression. In this study, oncolytic activity in vitro and antitumor therapeutic efficacy in vivo when combined with oHSV and panobinostat were investigated. (1) Treatment with panobinostat enhanced oHSV propagation and cytotoxicity in human glioma A172 and squamous cell carcinoma SCC9 cells. (2) Combined treatment with oHSV and panobinostat enhanced virus replication mediated by the transcriptional downregulation of IFN-β- and IFN-responsive antiviral genes in human glioma A172 and squamous cell carcinoma SCC9 cells. (3) Panobinostat treatment induced upregulation of PD-L1 expression in both glioma and squamous cell carcinoma cells. (4) A significantly enhanced therapeutic efficacy was shown in vivo for the murine glioma CT-2A and squamous cell carcinoma SCC7 models when treated with a combination of oHSV, including PD-1/PD-L1 blockade and HDAC inhibition. Consequently, these data provide some new clues for the clinical development of combination therapy with OVs, epigenetic modifiers, and checkpoint blockades for glioma and squamous cell carcinoma.

Metformin enhances the antitumor activity of oncolytic herpes simplex virus HF10 (canerpaturev) in a pancreatic cell cancer subcutaneous model

Oncolytic virus (OV) therapy is a promising cancer immunotherapy, especially for cold tumors by inducing the direct lysis of cancer cells and initiation of potent antitumor response. Canerpaturev (C-REV) is an attenuated oncolytic herpes simplex virus-1, which demonstrated a potent antitumor effect in various preclinical models when used either alone or combined. Metformin is a commonly prescribed antidiabetic drug that demonstrated a potent immune modulator effect and antitumor response. We combined C-REV with metformin in a low immunogenic bilateral murine tumor model to enhance C-REV’s antitumor efficacy. In vitro, metformin does not enhance the C-REV cell cytotoxic effect. However, in in vivo model, intratumoral administration of C-REV with the systemic administration of metformin led to synergistic antitumor effect on both sides of tumor and prolonged survival. Moreover, combination therapy increased the effector CD44+ CD8+ PD1- subset and decreased the proportion of terminally-differentiated CD103+ KLRG-1+ T-regulatory cells on both sides of tumor. Interestingly, combination therapy efficiently modulates conventional dendritic cells type-1 (cDC1) on tumors, and tumor-drained lymph nodes. Our findings suggest that combination of C-REV and metformin enhances systemic antitumor immunity. This study may provide insights into the mechanism of action of OV therapy plus metformin combination against various tumor models.

Overcoming resistance of stroma-rich pancreatic cancer with focal adhesion kinase inhibitor combined with G47Δ and immune checkpoint inhibitors

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease known for its dense tumor stroma. Focal adhesion kinase inhibitor (FAKi), a non-receptor type tyrosine kinase inhibitor, reduces the tumor stroma. G47Δ, a third-generation oncolytic herpes simplex virus type 1, destroys tumor cells selectively and induces antitumor immune responses. This study evaluates the efficacy of FAKi and G47Δ in PDAC models in combination with or without immune checkpoint inhibitors. G47Δ was effective in human PDAC cell lines invitro and in subcutaneous as well as orthotopic tumor models. Transgenic mouse-derived #146 cells were used to generate subcutaneous PDAC tumors with rich stroma in immunocompetent mice. In this #146 tumor model, the efficacy of FAKi was synergistically augmented when combined with G47Δ, which reflected not only a decreased stromal content but also a significant shifting of the tumor microenvironment toward immune stimulation. In transgenic autochthonous PKF mice, a rare model that develops stroma-rich PDAC with a 100% penetrance and resembles human PDAC in various aspects, the prolongation of survival compared with FAKi alone was achieved only when FAKi was combined with G47Δ and immune checkpoint inhibitors. The FAKi combination therapy may be useful to overcome the treatment resistance of stroma-rich PDAC.

TMIC-39. TARGETING EXTRACELLULAR MATRIX HYALURONIC ACID-CD44 SIGNALING REDUCES TUMOR STEMNESS AND SENSITIZES TUMOR TO VIROTHERAPY AND ENHANCES THERAPEUTIC POTENTIAL FOR CANCER TREATMENT

Abstract The tumor microenvironment (TME), including the non-tumor cells and the extracellular matrix (ECM) plays a crucial role in tumor progression and metastasis. Hyaluronic acid (HA), the major glycosaminoglycan present in brain ECM, has long been associated with the progression and invasiveness of brain tumors. HA signals primarily thorough CD44, an adhesion/homing receptor leading to the induction of cellular AKT, MEK and HIF signaling, thereby promoting tumor cell proliferation, aggressiveness and therapy resistance. While HA in the ECM has been shown to interfere with infection and spread of oncolytic viruses, the impact of tumor-ECM interaction induced signaling on oncolytic virotherapy is heavily understudied. RNA sequencing and gene set enrichment analysis of glioma cells infected with an oncolytic Herpes Simplex Virus-1 (oHSV) demonstrate an enrichment of pathways related to tumor-ECM interaction. Immunostaining of brain sections from intracranial tumor bearing mice also reveals increased HA after oHSV treatment. Our results further demonstrate that HA/CD44-mediated signaling inhibits virus replication in vitro. Herein, to evaluate the impact of blocking tumor-ECM interactions without altering the secreted ECM, we created oHSV-sCD44, an oHSV that encodes for extracellular soluble CD44 (sCD44) that can function as a dominant negative receptor for membrane bound CD44. oHSV-sCD44 significantly reduces the stemness of glioblastoma stem cells (GSCs), induces DNA damage and sensitizes the GSCs to radiation therapy. Intra-tumoral injection of oHSV-sCD44 into patient-derived primary GBM xenograft model significantly inhibits tumor growth accompanied by reduced stemness and decreased HA expression, and increased oHSV replication and tumor cell lysis in TME. Moreover, blocking HA-CD44 signaling with a single dose of oHSV-sCD44 in murine glioma syngeneic model induces the development of a significant anti-tumor immune response with enhanced T cell infiltration. Collectively, our findings implicate oHSV-sCD44 as a potential oncolytic and immune-stimulating anticancer therapeutic.

EXTH-42. DUAL ROLES OF PKR ORCHESTRATE ONCOLYTIC HSV SENSITIVITY AND ANTIGEN-SPECIFIC T CELL EXPANSION

Abstract The host immune system has developed many mechanisms to defend against and clear virus infections, including PKR, cGAS-STING and TLR-MyD88 signaling pathways. Compromised anti-viral mechanisms in tumor cells allows for infection, replication and lysis by oncolytic viruses. However, compromised antiviral mechanisms, together with immune cells and the tumor microenvironment (TME), limit the sensitivity of oncolytic virus in tumor cells. We generated a novel oncolytic Herpes simplex virus, oHSV-shPKR which disables tumor-intrinsic PKR signaling. oHSV-shPKR significantly increases oHSV infection and lysis in both oncolytic virus-resistant and sensitive glioblastomas (GBMs). Infection of oHSV-shPKR in GBMs induces G2/M cell cycle arrest and inhibits tumor cell growth. oHSV-shPKR increases activation of antigen presentation cells through type I interferon signaling activation and its immune-stimulatory function to increase tumor-antigen specific CD8 T-cell expansion, including cytotoxic T lymphocytes. Preclinical studies showed that oHSV-shPKR intra-tumoral injection significantly inhibits human GBM patient derived xenograft (PDX) tumor growth in immune-deficient NSG mice and murine orthotopic GBM tumor growth in immunocompetent mice. The results demonstrate that the novel oHSV-shPKR has the potential to be used in clinical applications for both oHSV-resistant and sensitive GBM treatment.

EXTH-51. HSV-PTENΑ TREATMENT TARGETS GLIOMA STEM CELLS TO REDUCE STEMNESS AND SENSITIZES GLIOBLASTOMA TO IRRADIATION

Abstract Glioblastoma is one of the most lethal and treatment-resistant tumors of the central nervous system. Despite maximal surgical and medical therapy, survival remains dismal with a median of 21 months. Despite advances in treatment, this has only led to modest survival benefit. A significant challenge in treatment of glioblastoma is targeting glioma stem cells (GSCs) which are a source of therapy resistance. Oncolytic viral (OV) therapy is a promising therapy for solid tumors that preferentially targets tumor cells for lysis and an anti-tumor immune response while sparing normal cells. Among all OVs, oncolytic Herpes Simplex Virus (oHSV) is substantially ahead in the clinic, with an oHSV T-VEC approved by the FDA for metastatic melanoma treatment and G47∆ which received conditional approval for the treatment of GBM in Japan. In prior work, our group has demonstrated that PTENα expression by an oHSV (HSV-P10) results in improved long-term survivors in intracranial tumor-bearing mice compared to HSVQ treatment. We aim to elucidate the mechanism of improved therapeutic efficacy of HSV-P10 against GBM and evaluate if HSV-P10 may overcome radio-resistance. RNA sequencing and GSEA analysis of primary human GBM cells infected with control HSVQ or HSV-P10 reveals that while HSVQ virus infection leads to an increase in genes regulating IL6-STAT3 pathway, pivotal in maintaining stemness properties, HSV-P10 infection leads to a reduction. HSV-P10 reduces CD133+/CD44+ stem cells, induces DNA damage and sensitizes the GBM cells to irradiation. Our findings reveal a novel mechanism induced by HSV-P10 in combination with irradiation where HSV-P10 modulates IL6-STAT3 signaling downregulating genes associated with stemness (Nestin, Sox2). HSV-P10 infection in combination with irradiation reduces GSC tumor sphere formation in vitro and sensitizes GBMs to radiotherapy in an intracranial mouse xenograft model. Our findings uncover a possible mechanism to overcome GSC-mediated therapy resistance to improve the therapeutic efficacy for GBM.

TMIC-04. TARGETING AN ECM PROTEIN INVOLVED IN BREAST CANCER BRAIN METASTASES WITH AN ONCOLYTIC HSV-1

Abstract The beta-1 subunit of integrins (CD29) is important for cell-extracellular matrix interactions. CD29 expression is increased in breast cancer cells, and it promotes their invasion, migration, and intravasation, all of which can result in breast cancer brain metastasis (BCBM). While CD29 expression can be correlated to poor prognosis of breast cancer patients, the median survival of BCBM patients predicted to have better prognosis is 36 months. Despite current BCBM treatments such as craniotomy, reaching the metastatic site is often challenging. Modified viral vectors, such as oncolytic herpes simplex virus (oHSV), have been clinically approved for the treatment of malignant melanoma and brain tumors, as they infect and selectively kill tumor cells. Previously, we identified that combination of oHSV and humanized CD29 blocking antibody (OS2966, aCD29) enhanced viral replication by reducing antiviral immune response, increased tumor cell killing, and rendered survival benefit to tumor bearing mice with intra-tumoral, but not systemic, oHSV administration. These findings suggested that the antitumor efficacy of the therapeutic combination required intratumoral delivery of anti-CD29, since the antibody could not penetrate the blood brain barrier and reach intracranial tumors. To overcome this, we report the generation of an oHSV encoding the scFv-Fc fragment of this antibody (HSV-aCD29), which retains its oncolytic function, and evaluate the impact of HSV-aCD29 on the CD29 downstream signaling. We will further characterize the safety, antitumor efficacy, and assess the immune response to HSV-aCD29 in vitro and in vivo. We anticipate that compared to our control virus, HSV-aCD29 will block CD29 activity which will reduce antiviral immune responses and enhance tumor cell lysis, thus improving oHSV efficacy. Together, this study proposes the blockade of CD29 with the targeted delivery of a CD29 blocking antibody mediated by a novel oHSV as a potential synergistic therapy against BCBM.

Targeting proteasome enhances anticancer activity of oncolytic HSV-1 in colorectal cancer

Herpes simplex virus 1 (HSV-1) has been widely used to treat various cancers, but its efficacy is limited. Studies indicated that combining HSV-1 and chemotherapy drugs can effectively improve the lethality of HSV-1 in tumor cells, which has a synergistic effect. Here, we explored the oncolytic effect and mechanism of bortezomib and HSV-1 on colorectal cancer cells, HCT116 and Caco-2. First, we selected four drugs to detect cell viability and found that the strongest HSV-1-promoting effect was achieved using bortezomib + HSV-1 treatment. Bortezomib combined with HSV-1 treatment significantly upregulated the expression of heat shock proteins, endoplasmic reticulum stress-related proteins and apoptosis-related proteins, while Bcl-2 was downregulated. JC-1 staining revealed that combining bortezomib and HSV-1 promotes cell apoptosis. In addition, bortezomib + oHSV-1 treatment effectively inhibit tumor growth. These results indicate that bortezomib combined with HSV-1 induce intense endoplasmic reticulum stress and activate the caspase-12 apoptosis pathway, killing tumor cells.

Specific targeting of glioblastoma with an oncolytic virus expressing a cetuximab-CCL5 fusion protein via innate and adaptive immunity.

Chemokines such as C-C motif ligand 5 (CCL5) regulate immune cell trafficking in the tumor microenvironment (TME) and govern tumor development, making them promising targets for cancer therapy. However, short half-lives and toxic off-target effects limit their application. Oncolytic viruses (OVs) have become attractive therapeutic agents. Here, we generate an oncolytic herpes simplex virus type 1 (oHSV) expressing a secretable single-chain variable fragment of the epidermal growth factor receptor (EGFR) antibody cetuximab linked to CCL5 by an Fc knob-into-hole strategy that produces heterodimers (OV-Cmab-CCL5). OV-Cmab-CCL5 permits continuous production of CCL5 in the TME, as it is redirected to EGFR+ glioblastoma (GBM) tumor cells. OV-Cmab-CCL5 infection of GBM significantly enhances the migration and activation of natural killer cells, macrophages and T cells; inhibits tumor EGFR signaling; reduces tumor size; and prolongs survival of GBM-bearing mice. Collectively, our data demonstrate that OV-Cmab-CCL5 offers a promising approach to improve OV therapy for solid tumors.

SIRPα antibody combined with oncolytic virus OH2 protects against tumours by activating innate immunity and reprogramming the tumour immune microenvironment

BackgroundThe combination of oncolytic viruses (OVs) with immune checkpoint blockades is a research hotspot and has shown good efficacy. Here, we present the first attempt to combine oncolytic herpes simplex virus 2 (OH2) with an anti-SIRPα antibody as an antitumour treatment. Our results provide unique insight into the combination of innate immunity with OV.MethodsWe verified the polarization and activation of OH2 in RAW264.7 cells in vitro. Subsequently, we evaluated the antitumour ability of OH2 and anti-SIRPα combined therapy in a tumour-bearing mouse model. RNA-seq and Single-cell RNA-seq were used to characterize the changes in the tumour microenvironment.ResultsThe OH2 lysates effectively stimulated RAW264.7 cells to polarize towards the M1 but not the M2 phenotype and activated the function of the M1 phenotype in vitro. In the macrophage clearance experiment, OH2 therapy induced polarization of M1 macrophages and participated in the antitumour immune response in a tumour-bearing mouse model. Treatment with a combination of OH2 and anti-SIRPα effectively inhibited tumour growth and significantly prolonged the survival time of the mice, and this result was more obvious in the mouse model with a larger tumour volume at the beginning of the treatment. These results suggest that combination therapy can more profoundly reshape the TME and activate stronger innate and adaptive immune responses.ConclusionsOur data support the feasibility of oncolytic virus therapy in combination with anti-SIRPα antibodies and suggest a new strategy for oncolytic virus therapy.

Oncolytic herpes simplex virus delivery of dual CAR targets of CD19 and BCMA as well as immunomodulators to enhance therapeutic efficacy in solid tumors combined with CAR T cell therapy.

The CAR T-cell therapy is a promising approach to treating hematologic malignancies. However, the application in solid tumors still has many tough challenges, including heterogenicity in antigen expressions and immunosuppressive tumor microenvironment (TME). As a new cancer treatment modality, oncolytic virotherapy can be engineered to circumvent these obstacles for CAR T cell therapy in solid tumors. In this study, an oHSV T7011 is engineered to drive ectopic expression of dual-antigens, extracellular domains of CD19 and BCMA, on the solid tumor cell surface to be targeted by approved CAR T cells. In addition, multiple immunomodulators, CCL5, IL-12, and anti-PD-1 antibody are also included to modulate the TME. The antitumor activities of T7011 in combination with CD19 or BCMA CAR T-cell were evaluated in vitro and in vivo. The expression of CD19 or BMCA on the tumor cell surface could be detected after T7011 infection. The level of CCL5 in TME was also increased. Efficacy studies demonstrated that combination with T7011 and CAR-TCD19 or CAR-TBCMA cells showed significant synergistic anti-tumor responses in several solid tumor models. These studies indicated that the new generation of oHSV T7011 can be a promising combinational therapy with CD19 or BCMA-specific CAR T cells for the treatment of a broad range of solid tumors.

Construction of an IL12 and CXCL11 armed oncolytic herpes simplex virus using the CRISPR/Cas9 system for colon cancer treatment

Oncolytic viruses are an emerging cancer treatment modality with promising results in clinical trials. The new generation of oncolytic viruses are genetically modified to enhance virus selectivity for tumor cells and allow local expression of therapeutic genes in tumors. The traditional technique for viral genome engineering based on homologous recombination using a bacterial artificial chromosome (BAC) system is laborious and time-consuming. With the advent of the CRISPR/Cas9 system, the efficiency of gene editing in human cells and other organisms has dramatically increased. In this report, we successfully applied the CRISPR/Cas9 technique to construct an HSV-based oncolytic virus, where the ICP34.5 coding region was replaced with the therapeutic genes murine interleukin 12 (IL12, p40-p35) and C-X-C motif chemokine ligand 11 (CXCL11), and ICP47 gene was deleted. The combination of IL12 and CXCL11 in oncolytic viruses showed considerable promise in colorectal cancer (CRC) treatment. Overall, our study describes genetic modification of the HSV-1 genome using the CRISPR/Cas9 system and provides evidence from principle studies for engineering of the HSV genome to express foreign genes.

Oncolytic Herpes Simplex Virotherapy for Pediatric Neuroendocrine Cancer

INTRODUCTION: Therapeutic options remain limited for children with gastroenteropancreatic neuroendocrine tumors (giNET). We have previously shown that M002, an oncolytic Herpes simplex virus, effectively killed cells from a pediatric giNET patient-derived xenograft (PDX). We aimed to corroborate and validate these findings using an established neuroendocrine cell line, QGP-1, for further in vitro investigations, and a human giNET PDX for in vivo studies. METHODS: Cell surface expression of the viral entry receptors (CD111, CD112, Syndecan, and herpesvirus entry mediator [HVEM]) was detected by flow cytometry. Single-step viral recovery assays assessed viral replication. Cytotoxicity was determined via alamarBlue assay. PDX cells (1x106) were subcutaneously injected into the flanks of athymic nude mice and resulting tumors were treated with a single intratumoral injection of M002 (107 plaque forming units [PFU]). RESULTS: QGP-1 cells expressed each viral entry receptor on its cell surface (CD111 = 50%, CD112 = 34%, syndecan = 36%, and HVEM = 71%; Fig. 1A). M002 replicated in the QGP-1 cells with viral titer increasing over 12 and 24 hours (3.8E7 PFU/mL, 1.5E14 PFU/mL, respectively). M002 treatment significantly decreased QGP-1 viability (Fig. 1B). Tumors treated with M002 were significantly smaller than vehicle-treated tumors, and M002 treatment significantly prolonged animal survival (Figs. 1C, 1D).CONCLUSION: M002 successfully infects, replicates within, and kills QGP-1 cells. In an established PDX in vivo model, M002 treatment delayed tumor growth and increased animal survival. These data provide preclinical data supporting translation of oncolytic virotherapy for pediatric giNET tumors to the clinical setting.

Safety and Efficacy of Intraventricular Immunovirotherapy with Oncolytic HSV-1 for CNS Cancers.

Oncolytic virotherapy with herpes simplex virus-1 (HSV) has shown promise for the treatment of pediatric and adult brain tumors; however, completed and ongoing clinical trials have utilized intratumoral/peritumoral oncolytic HSV (oHSV) inoculation due to intraventricular/intrathecal toxicity concerns. Intratumoral delivery requires an invasive neurosurgical procedure, limits repeat injections, and precludes direct targeting of metastatic and leptomeningeal disease. To address these limitations, we determined causes of toxicity from intraventricular oHSV and established methods for mitigating toxicity to treat disseminated brain tumors in mice. HSV-sensitive CBA/J mice received intraventricular vehicle, inactivated oHSV, or treatment doses (1×107 plaque-forming units) of oHSV, and toxicity was assessed by weight loss and IHC. Protective strategies to reduce oHSV toxicity, including intraventricular low-dose oHSV or interferon inducer polyinosinic-polycytidylic acid (poly I:C) prior to oHSV treatment dose, were evaluated and then utilized to assess intraventricular oHSV treatment of multiple models of disseminated CNS disease. A standard treatment dose of intraventricular oHSV damaged ependymal cells via virus replication and induction of CD8+ T cells, whereas vehicle or inactivated virus resulted in no toxicity. Subsequent doses of intraventricular oHSV caused little additional toxicity. Interferon induction with phosphorylation of eukaryotic initiation factor-2α (eIF2α) via intraventricular pretreatment with low-dose oHSV or poly I:C mitigated ependyma toxicity. This approach enabled the safe delivery of multiple treatment doses of clinically relevant oHSV G207 and prolonged survival in disseminated brain tumor models. Toxicity from intraventricular oHSV can be mitigated, resulting in therapeutic benefit. These data support the clinical translation of intraventricular G207.

Histone deacetylase inhibitors enhance oncolytic herpes simplex virus therapy for malignant meningioma

Approximately 20% of meningiomas are not benign (higher grade) and tend to relapse after surgery and radiation therapy. Malignant (anaplastic) meningioma (MM) is a minor subset of high-grade meningioma that is lethal with no effective treatment options currently. Oncolytic herpes simplex virus (oHSV) is a powerful anti-cancer modality that induces both direct cell death and anti-tumor immunity, and has shown activity in preclinical models of MM. However, clinically meaningful efficacy will likely entail rational mechanistic combination approaches. We here show that epigenome modulator histone deacetylase inhibitors (HDACi) increase anti-cancer effects of oHSV in human MM models, IOMM-Lee (NF2 wild-type) and CH157 (NF2 mutant). Minimally toxic, sub-micromolar concentrations of pan-HDACi, Trichostatin A and Panobinostat, substantively increased the infectability and spread of oHSV G47Δ within MM cells in vitro, resulting in enhanced oHSV-mediated killing of target cells when infected at low multiplicity of infection (MOI). Transcriptomics analysis identified selective alteration of mRNA processing and splicing modules that might underlie the potent anti-MM effects of combining HDACi and oHSV. In vivo, HDACi treatment increased intratumoral oHSV replication and boosted the capacity of oHSV to control the growth of human MM xenografts. Thus, our work supports further translational development of the combination approach employing HDACi and oHSV for the treatment of MM.

VG161 activates systemic antitumor immunity in pancreatic cancer models as a novel oncolytic herpesvirus expressing multiple immunomodulatory transgenes.

The VG161 represents the first recombinant oncolytic herpes simplex virus type 1 carrying multiple synergistic antitumor immuno-modulating factors. Here, we report its antitumor mechanisms and thus provide firm theoretical foundation for the upcoming clinical application in pancreatic cancer. Generally, the VG161-mediated antitumor outcomes were analyzed by a collaboration of techniques, namely the single-cell sequencing, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADSI-MSI) and nanostring techniques. In vitro, the efficacy of VG161 together with immune checkpoint inhibitors (ICIs) has been successfully shown to grant a long-term antitumor effect by altering tumor immunity and remodeling tumor microenvironment (TME) metabolisms. Cellular functional pathways and cell subtypes detected from patient samples before and after the treatment had undergone distinctive changes including upregulated CD8+ Tand natural killer cells. More importantly, significant antitumor signals have emerged since the administration of VG161 injection. In conclusion, VG161 can systematically activate acquired and innate immunity in pancreatic models, as well as improve the tumor immune microenvironment, indicative of strong antitumor potential. The more robusting antitumor outcome for VG161 monotherapy or in combination with other therapies on pancreatic cancer is worth of being explored in further clinical trials.

Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma.

Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR]domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.