Oncolytic Mutant Research Articles

624. Effect of an Oncolytic HSV Mutant Expressing ICP34.5 under Control of a Nestin Promoter for Brain Tumor Therapy

HSV mutants (e.g. G207 and MGH1), possessing deletions in both copies of the ICP34.5 gene and an insertional mutation in the ICP6 gene (ribonucleotide reductase), have proven safe through a number of animal experiments and clinical trials. Although these double mutants clearly demonstrate oncolytic effects as well as anti-tumor immunization, their oncolytic effects are markedly reduced. ICP34.5-null viruses fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. In HSV-infected cells, viral double-stranded RNA will activate PKR by autophosphorylation, and activated PKR phosphorylates eIF-2a, which inhibits initiation of protein translation within the cells and in turn inhibits viral replication. The ICP34.5 dephosphorylates eIF-2a, thus allowing continued protein synthesis and viral replication. In order to increase therapeutic efficacy and tumor selectivity, glioma selective expression of ICP34.5 in oncolytic HSV has been explored. As a candidate for glioma selective promoters, we examined a synthetic nestin promoter, which consists of the nestin enhancer and hsp68 minimum promoter. We first performed RT-PCR to examine nestin expression in various types of cultured cells. All the glioma cell lines tested (U251, U87dEGFR, Gli36d5, T98G, MGH238, U138) were confirmed to be positive for nestin expression. Astrocytes, on the other hand, were negative. We, therefore, constructed a novel HSV mutant, rQNestin34.5, which expresses ICP34.5 under control of the synthetic nestin promoter. In the experiments using cultured human glioma cell lines in vitro, rQNestin34.5 showed remarkable enhancements in both viral propagation and oncolysis compared to the ICP34.5-null virus (rHsvQ1). In four primary glioblastoma cells, rQNestin34.5 also showed remarkable enhancement in both viral propagation and oncolysis compared to control rHsvQ1. In normal human astrocytes, rQNestin34.5 showed equivalent toxicity to control rHsvQ1. We also performed Western blotting to examine the phosphorylation state of eIF2a. In glioma cell lines infected with rQNestin34.5, the level of phspho-eIF2a was lower than that of control rHsvQ1, confirming that the ICP34.5 is expressed selectively in glioma cells and resulted in a decrease of phospho-eIF2a level. This was responsible for its efficient propagation in glioma cells. We further characterized the anti-cancer efficacy of this new virus in vivo using an athymic mouse subcutaneous tumor model (U87dEGFR cells) and a brain tumor model (U87dEGFR cells). The rQNestin34.5 showed significantly more potent inhibition of tumor growth compared to control rHsvQ1. The mice with brain tumor treated by rQNestin34.5 survived longer than those treated by rHsvQ1.

979. The Gene Expression and Function of Adenoviral E3-Genes in Murine Tumour Cell Lines

We recently developed a number of immunocompetent murine tumour models that enabled the evaluation of adenoviruses with classical E3 deletions, dl309 (E3B deleted (RIDa/RIDb complex and 14.7kD)) and dl704 (E3 gp19K deleted). The E3B genes protect infected cells from tumour necrosis factor (TNF) and Fas mediated apoptosis, and internalizes epidermal growth factor receptor (EGFR). In addition, E3 gp19K prevents newly synthesised major histocompatibility complex (MHC) class 1 molecules from reaching the cell surface, thus avoiding immune recognition of infected cells. Three murine tumour cell lines (CMT64, JC and CMT93) and two human control tumour cell lines (A549 and H460) were characterised in vitro after infection with Ad5, (wild type E3), dl309, dl704 and dl312 (E1A deleted, wild type E3). The dl309 mutant was more potent compared to Ad5 in each cell line tested while dl704 was less potent than Ad5. Compared with Ad5, dl309 viral replication was higher in every cell line at each time point tested while dl704 replication was lower than Ad5. In one murine cell line (CMT64), viral burst was similar to the observed levels in the human H460 cells while two cell lines (JC and CMT93) had lower or no replication. Adenoviral E3 gene expression (E3B and gp19K) was supported in each murine tumour line. In addition, the E3 proteins had similar function in the murine cell lines as previously reported for human cell lines. We demonstrated EGFR, Fas and TNFR1 internalisation/degradation following Ad5 and dl704 infection while Ad5 and dl309 infection resulted in loss of extracellular MHC class 1 receptor expression/detection. The decrease in MHC class 1 cell surface expression was not observed following dl704 infection. These results strengthen our previous findings using murine immunocompetent xenograft models in the evaluation of interactions between the host immune system and tumour cells infected with oncolytic viral mutants. The further development of these murine tumour models will aid in the improved design of more efficacious E3-deletion mutants.

62. Ras Signaling as a Marker for Application of Oncolytic Herpes Mutant Against Lymphoma

Herpes simplex virus (HSV) enters cells via initial binding of envelope glycoproteins C and B to cell-surface glycosaminoglycans (GAGs) and subsequent membrane fusion involving gD, gB and gH/gL. Current insights suggest that the fusion process is initiated by interaction of gD with a cognate cellular receptor, such as the widely-distributed cell adhesion molecule nectin-1/CD115. To re-direct the tropism of HSV-1, we have generated a soluble adapter protein comprising the gD-binding variable (V) domain of nectin-1 fused to a single-chain antibody (V528LH) recognizing the EGF receptor. The adapter molecule enabled HSV-1 entry into naturally non-permissive CHO cells expressing the EGF receptor, but not into CHO cells lacking the receptor. Furthermore, entry was not observed when the antibody portion of the adapter was replaced with an antibody of different specificity. The degree of P-V528LH-adapter-mediated entry increased with the viral dose, reaching approximately 65% infected cells at a multiplicity of 3. Entry depended on viral gD and the availability of cellular GAGs. Although the adapter molecule did not block nectin-1-mediated cellular susceptibility to HSV-1, preconditioning of nectin-1-bearing cells with competitive inhibitors of the normal nectin-1/gD interaction reduced nectin-1-mediated entry without limiting adapter-mediated entry via the EGF receptor. These results indicate that an adapter molecule along with nectin-1 blocking agents can re-direct HSV infection.

608. VAI Deleted Adenovirus as an Oncolytic Agent Targeting Human EBV-Associated Tumours

The EBV genome is frequently present in Burkitt's lymphomas, B-Cell lymphomas of immunocompromised patients, nasopharyngeal carcinomas, Hodgkin's disease, T-cell lymphomas, natural killer cell leukemia/lymphoma, smooth-muscle tumors and gastric cancer. More recently, there have been scattered reports linking EBV with conventional epithelial cancers of other primary sites including breast cancers, lung cancers, prostate cancers, liver cancers, colon cancers and lymphoepithelioma-like carcinoma of the esophagus. Given the ever-growing number of tumor types associated with EBV infection, it is therefore critically important to develop therapeutic strategies to treat EBV-associated tumors. Replication-selective oncolytic adenovirus is a promising new platform for anti-cancer therapy. Data from numerous clinical trials have demonstrated several attenuated oncolytic viral mutants to be safe and to replicate selectively in tumor cells with significant anti-tumor potency. Virus associated (VA) 1 and 2 RNA are two low molecular weight adenoviral RNAs that are synthesized late during the viral replication cycle and are required for efficient translation of late viral mRNAs. When the VA1 gene is deleted, replication is impeded in most cells (including HEK 293 cells). The Epstein Barr Virus (EBV) genome encodes two small, non-polyadenylated nuclear RNAs (EBER 1 and 2) that are uniformly expressed in most EBV-associated human tumors. EBER 1 and 2 can functionally substitute for the VA1 RNAs of adenovirus, enabling replication to proceed normally through complementation of VA1 deletion mutants. We hypothesized that VA1-deleted adenovirus would selectively replicate in EBV-positive tumor cells due to complementation by EBER 1, with poor or no replication in normal cells and EBV-negative tumor cells. Our data demonstrate that cytotoxicity of VA1 RNA deletion mutants was restricted to EBV-positive tumor cells (e.g. c666, AGS-EBV, Raji, Jiyoye). Cytopathic effects were much lower in EBV-negative tumor cells (Hep-2, AGS-ATCC, Panc-1). The replication of the VA1 deletion mutant virus was 14–25 times higher in EBV-positive tumor cells than in EBV-negative tumor cells and comparable with wild type adenovirus. The VA1 mutant did not replicate in normal cells in culture such as normal human bronchial epithelial cells (NHBE), and replicated poorly in prostate epithelial cells (PrEC). In vivo, VAI deleted adenovirus showed the same efficacy as wild type adenovirus in xenograft models of human EBV-positive tumor cells, but much lower cytotoxicity than wildtype adenovirus. Our data suggest that a VA1 RNA-deleted viral mutant is a promising replication-selective oncolytic virus specifically targeting EBV-associated cancers.

Infection with oncolytic herpes simplex virus-1 induces apoptosis in neighboring human cancer cells: a potential target to increase anticancer activity.

The antitumor efficacy of a herpes simplex virus (HSV)-1 oncolytic virus depends on the cytotoxic effect of the virus, but also on viral replication and spread within the tumor. Apoptosis is considered a defense mechanism of infected cells that minimizes the spread of viral progeny by limiting cellular production of virus. We sought to determine whether oncolytic HSV-1 infection induces apoptosis in neighboring, uninfected cells and whether manipulation of apoptosis can increase viral replication and cytotoxicity. NV1066 is an oncolytic HSV-1 mutant that contains the marker gene for enhanced green fluorescent protein. OCUM human gastric cancer cells were infected with NV1066 in vitro and inspected for apoptosis by Hoechst and terminal deoxynucleotidyltransferase-mediated nick end labeling staining and for infection by expression of green fluorescence. A significant increase in apoptosis was seen in cells infected by NV1066. More interestingly, a significant percentage (10%) of uninfected cells also proceeded to apoptosis. After NV1066 infection, cells were also treated with N-acetylcysteine (NAC), an inhibitor of apoptosis. By day 4 after infection, 2.7x more NV1066 was produced in cells exposed to NAC than in those not exposed to NV1066 (P = 0.04). NAC also increased tumor kill when administered with virus. These data suggest that NV1066 induces apoptosis in uninfected cocultured cells, potentially hindering propagation of viral progeny and concomitant tumor kill. Inhibition of apoptosis may improve the efficacy of oncolytic HSV-1 therapy.

Oncogenes in Ras signalling pathway dictate host-cell permissiveness to herpes simplex virus 1.

The importance of herpes simplex viruses (HSV) as human pathogens and the emerging prospect of using mutant derivatives of HSV-1 as potential anti-cancer therapeutics have necessitated a thorough investigation into the molecular basis of host-cell permissiveness to HSV. Here we show that NIH-3T3 cells transformed with the oncogenes v-erbB, activated sos or activated ras become significantly more permissive to HSV-1. Inhibitors of the Ras signalling pathway, such as farnesyl transferase inhibitor 1 and PD98059, effectively suppressed HSV-1 infection of ras-transformed cells. Enhanced permissiveness of the transformed cells was linked to the inhibition of virus-induced activation (phosphorylation) of the double-stranded RNA-activated protein kinase (PKR), thereby allowing viral transcripts to be translated in these cells. An HSV-1-derived oncolytic mutant, R3616, was also found to infect preferentially both transformed cells and PKR-/- (but not PKR+/+) mouse embryo fibroblasts. These observations suggest that HSV-1 specifically targets cells with an activated Ras signalling pathway, and have important ramifications in the use of engineered HSV in cancer therapy, the development of strategies against HSV infections, and the controversial role of HSV in human cancers.

An oncolytic viral mutant that delivers the CYP2B1 transgene and augments cyclophosphamide chemotherapy.

Herpes simplex viruses type 1 (HSV-1) with an inactivated viral ribonucleotide reductase (Hsrr, ICP6) were designed to target tumor cells with upregulated mammalian ribonucleotide reductase (mRR), an enzyme whose expression is regulated by the p16/pRB tumor suppressor pathway. A recombinant HSV-1 was generated by knock-out of Hsrr and insertion of the rat CYP2B1 transgene responsible for the bioactivation of the prodrugs, cyclophosphamide and ifosfamide. The mutant virus replicated selectively in rat and human tumor cells that express mRR. Addition of cyclophosphamide potentiated oncolytic effects against cultured tumor cells and subcutaneous tumor xenografts established in athymic mice.