Expression of RNA interference triggers from an oncolytic herpes simplex virus results in specific silencing in tumour cells in vitro and tumours in vivo.

BACKGROUND: Delivery of small interfering RNA (siRNA) to tumours remains a major obstacle for the development of RNA interference (RNAi)-based therapeutics. Following the promising pre-clinical and clinical results with the oncolytic herpes simplex virus (HSV) OncoVEX GM-CSF, we aimed to express RNAi triggers from oncolytic HSV, which although has the potential to improve treatment by silencing tumour-related genes, was not considered possible due to the highly oncolytic properties of HSV. METHODS: To evaluate RNAi-mediated silencing from an oncolytic HSV backbone, we developed novel replicating HSV vectors expressing short-hairpin RNA (shRNA) or artificial microRNA (miRNA) against the reporter genes green fluorescent protein (eGFP) and ß-galactosidase (lacZ). These vectors were tested in non-tumour cell lines in vitro and tumour cells that are moderately susceptible to HSV infection both in vitro and in mice xenografts in vivo. Silencing was assessed at the protein level by fluorescent microscopy, x-gal staining, enzyme activity assay, and western blotting. RESULTS: Our results demonstrate that it is possible to express shRNA and artificial miRNA from an oncolytic HSV backbone, which had not been previously investigated. Furthermore, oncolytic HSV-mediated delivery of RNAi triggers resulted in effective and specific silencing of targeted genes in tumour cells in vitro and tumours in vivo, with the viruses expressing artificial miRNA being comprehensibly more effective. CONCLUSIONS: This preliminary data provide the first demonstration of oncolytic HSV-mediated expression of shRNA or artificial miRNA and silencing of targeted genes in tumour cells in vitro and in vivo. The vectors developed in this study are being adapted to silence tumour-related genes in an ongoing study that aims to improve the effectiveness of oncolytic HSV treatment in tumours that are moderately susceptible to HSV infection and thus, potentially improve response rates seen in human clinical trials.

Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+.

BACKGROUND: Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). METHODS: To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. RESULTS: The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 "factory" cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. CONCLUSIONS: The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products.

Decreased replication ability of E1-deleted adenoviruses correlates with increased brain tumor malignancy.

E1 region replacement adenoviruses are replication defective and are propagated in cells providing adenovirus E1A and E1B proteins. Although they are being developed for antitumor therapies, the proliferative behaviors of these viruses in normal brain tissues or in brain tumors are unknown. To address this, freshly cultured cells from normal human brain and common brain tumors (astrocytomas and meningiomas) were infected using wild-type species C adenoviruses and adenoviruses missing E1A (H5dl312) or E1A plus E1B (H5dl434). Viral DNA replication, late viral protein expression, and production of infectious progeny were characterized. Wild-type adenoviruses grew efficiently in normal brain and brain tumor cells. In comparison, E1-deleted adenovirus DNA replication was delayed and lower in cells derived from normal brain tissues, meningiomas, and low-grade astrocytomas. However, in contrast, E1-deleted adenovirus DNA replication did not occur or was extremely low in cells derived from malignancy grade III and IV astrocytic tumors. Because wild-type adenoviruses infected and replicated in all cells, the malignancy grade-based differential E1-deleted adenovirus DNA replication was not explained by differential virus uptake. Infectious H5dl312 and H5dl434 production correlated with viral DNA replication. Compared with a 5-day average for wild-type infections, advanced cytopathology was noted approximately 4 weeks after H5dl312 or H5dl434 infection of meningioma, astrocytoma, and normal brain cells. Cytopathology was not observed after H5dl312 or H5dl434 infection of glioblastoma, anaplastic astrocytoma, and gliosarcoma cells. Because of this tumor grade-based differential growth, the E1-deleted adenoviruses may represent novel tools for studies of brain tumor malignancy.

The effect of ganciclovir on herpes simplex virus-mediated oncolysis.

Entry of herpes simplex virus (HSV) into tumor cells results in viral gene expression followed by cellular lysis. Attenuated HSVs selectively destroy tumors with sparing of surrounding normal tissue. HSV encodes a thymidine kinase (TK) that converts ganciclovir to a toxic metabolite. This metabolite may be transferred between cells and lead to the death of neighboring uninfected cells, termed bystanders. We sought to determine if HSV-mediated oncolysis is enhanced by ganciclovir treatment. In addition, we examined bystander killing in cocultures of TK transfectants and parental cells. hrR3, an attenuated HSV, expresses TK. The 50% lethal dose of hrR3 for a rat gliosarcoma (9L) and three human colorectal carcinomas (HT29, KM12C6, and KM12L4) was determined. Cells were infected with a 50% lethal dose of hrR3, followed by treatment with ganciclovir, and then cell survival was quantitated. In separate experiments 9L and HT29 cells were transfected with TK. Parental cells and TK transfectants were cocultured in various ratios, in the presence of ganciclovir, and cell survival was quantitated. hrR3-mediated oncolysis was enhanced by ganciclovir in the gliosarcoma but not in the three colorectal carcinomas. Cocultures of both 9L and HT29 parental cells with their corresponding TK transfectants demonstrated bystander killing. The mortality of 9L cocultures was 54% greater than that predicted for exclusive killing of transfectants. HT29 mortality was 8% greater than predicted. The ability of ganciclovir to augment hrR3-mediated oncolysis varies significantly between tumor cells lines. The extent of ganciclovir-mediated killing of neighboring nontransduced parental cells similarly varies. Consideration should be given to these factors in the design of gene therapy strategies using HSV vectors as oncolytic agents.

Antitumor activity and reporter gene transfer into rat brain neoplasms inoculated with herpes simplex virus vectors defective in thymidine kinase or ribonucleotide reductase.

Herpes simplex virus (HSV) mutants or recombinant vectors might be useful oncolytic agents. Three general types of HSV vectors can be potentially used for this purpose: (1) mutants in viral transcription factors, such as ICP0 and ICP4; (2) mutants in enzymes involved in nucleic acid metabolism, such as thymidine kinase (TK) and ribonucleotide reductase (RR); and (3) mutants in neurovirulence factors, such as gamma 34.5. We tested the destructive ability of each type against rat 9L gliosarcoma cells in culture. We found that the HSV vectors defective in TK or RR were more efficient at tumor cell lysis in culture than the other types of HSV vectors. This increased efficiency provided the rationale for evaluating the TK and RR mutants in vivo following their stereotactic inoculation into 9L gliosarcomas implanted in rat brains. We employed the X-gal enzymatic histochemical assay to show that HSV-mediated lacZ gene expression was present in cells within the tumor mass in a relatively selective fashion. Immunoreactive HSV capsid and core antigens were present both in cells within the tumor, as well as in cells such as neurons and astrocytes, directly adjacent to the tumor mass. Long-term survival studies revealed that rats treated with either the TK or RR mutant lived significantly longer than control rats (p = 0.014, Kruskal-Wallis one-way analysis of variance). These results indicate that HSV vectors, defective in enzymes needed in nucleic acid metabolism, can preferentially mediate lacZ gene expression in cells within the tumor. (ABSTRACT TRUNCATED AT 250 WORDS)