Systemic therapy for cervical cancer with potentially regulatable oncolytic adenoviruses

Clinical trials have confirmed the safety of selectively oncolytic adenoviruses for treatment of advanced cancers. However, increasingly effective viruses could result in more toxicity and therefore it would be useful if replication could be abrogated if necessary. We analyzed viruses containing the cyclooxygenase-2 (Cox-2) or vascular endothelial growth factor (VEGF) promoter for controlling replication. Anti-inflammatory agents can lower Cox-2 protein levels and therefore we hypothesized that also the promoter might be affected. As Cox-2 modulates expression of VEGF, also the VEGF promoter might be controllable. First, we evaluated the effect of anti-inflammatory agents on promoter activity or adenovirus infectivity in vitro. Further, we analyzed the oncolytic potency of the viruses in vitro and in vivo with and without the reagents. Moreover, the effect of on virus replication was analyzed. We found that RGD-4C or Ad5/3 modified fibers improved the oncolytic potency of the viruses in vitro and in vivo. We found that both promoters could be downregulated with dexamethasone, sodium salicylate, or salicylic acid. Oncolytic efficacy correlated with the promoter activity and in vitro virus production could be abrogated with the substances. In vivo, we saw good therapeutic efficacy of the viruses in a model of intravenous therapy of metastatic cervical cancer, but the inhibitory effect of dexamethasone was not strong enough to provide significant differences in a complex in vivo environment. Our results suggest that anti-inflammatory drugs may affect the replication of adenovirus, which might be relevant in case of replication associated side effects.     

Cancer cell death by design: Apoptosis,autophagy and glioma virotherapy

Autophagy has been defined as a mechanism by which oncolytic adenoviruses mediate cell killing in some cancers, including malignant glioma. Until recently, however, adenovirus replication was regarded as a process that induced classical apoptosis in the infected cell. We have assessed the method of conditionally replicating adenovirus (CRAd) death in a model of malignant glioma, considering both autophagy and apoptosis as possible mechanisms of virally-induced cell death. Our initial investigations indicated that autophagy was the predominant system in CRAd-induced cell death in glioma. This appeared to be the case in vitro; however, further investigation in vivo shows that CRAds are capable of inducing both apoptotic and autophagic cell death. In this punctum, we summarize our latest research to uncover the method of oncolytic adenovirus-induced cell death in malignant glioma. Elucidating the relationship between autophagy and apoptosis in glioma virotherapy has significant implications for the design of optimal viral vectors.     

Linked tumor-selective virus replication and transgene expression from E3-containing oncolytic adenoviruses

Historically, the adenoviral E3 region was found to be nonessential for viral replication in vitro. In addition, adenoviruses whose genome was more than approximately 105% the size of the native genome were inefficiently packaged. These profound observations were used experimentally to insert transgenes into the adenoviral backbone. More recently, however, the reintroduction of the E3 region into oncolytic adenoviruses has been found to positively influence antitumor efficacy in preclinical models and clinical trials. In the studies reported here, the granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA sequence has been substituted for the E3-gp19 gene in oncolytic adenoviruses that otherwise retained the E3 region. Five viruses that differed slightly in the method of transgene insertion were generated and compared to Ar6pAE2fGmF (E2F/GM/DeltaE3), a previously described E3-deleted oncolytic adenovirus encoding GM-CSF. In all of the viruses, the human E2F-1 promoter regulated E1A expression and GM-CSF expression was under the control of the adenoviral E3 promoter and the packaging signal was relocated immediately upstream from the right terminal repeat. The E3-gp19-deleted viruses had similar cytolytic properties, as measured in vitro by cytotoxicity assays, but differed markedly in their capacity to express and secrete GM-CSF. Ar15pAE2fGmF (E2F/GM/E3b), the virus that produced the highest levels of GM-CSF and retained the native GM-CSF leader sequence, was selected for further analysis. The E2F/GM/E3b and E2F/GM/DeltaE3 viruses exhibited similar cytotoxic activity and GM-CSF production in several tumor cell lines in vitro. However, when compared in vivo in nude mouse xenograft tumor models, E2F/GM/E3b spread through tumors to a greater extent, resulted in higher peak GM-CSF and total exposure levels in both tumor and serum, and was more efficacious than the E3-deleted virus. Using the matched WI-38 (parental) and WI-38-VA13 (simian virus 40 large T antigen transformed) cell pair, GM-CSF was shown to be selectively produced in cells expressing high levels of E2F, indicating that the tumor-selective E2F promoter controlled E1A and GM-CSF expression.     

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.     

Therapy of cancer by cytokines mediated by gene therapy approach

Gene therapy offers a new approach for treatment of cancer. Transfer of genes encoding immunostimulatory cytokines has been used with remarkable success to eliminate cancer in animals. However, clinical trials in patients with this strategy had limited efficacy. Therefore, improvement ofgene transfer vector system is necessary. A hybrid viral vector, consisting of SFV replicon with either murine IL-12 or reporter LacZ gene, was constructed. This hybrid vector showed specificity and high level of expression in HCC both in vitro and in vivo. In a rat orthotropic liver tumor model, treatment of established tumors by the hybrid vector with raiL- 12 gene resulted in a strong anti-tumor activity without accompanying toxicity. Subsequently, a helper-dependent adenovirus vectors containing a mifepristone （RU486） inducible system was constructed for controlled and liver-specific expression of human interleukin 12 （hIL- 12） （HD-Ad/RUhIL- 12） and mouse IL-12 （mIL-12） （HD-Ad/RUmIL-12）. Data showed that high and sustained serum levels of hlL-12 could be attained by continuing administration of RU486 every 12 or 24 h. Repetitive induction ofhlL-12 could be obtained over, at least, a period of 48 weeks after a single injection of HD-Ad/RUhlL-12. Treatment of liver metastases with of HD-Ad/RUmIL- 12 plus RU846 resulted in complete tumor regression in all animals. Then, different cytokine genes were inserted into conditional replicative adenoviruses vectors （also called oncolytic adenovirus）. Replication ofadenovirus in tumor cells would kill tumor cells and release viruses, which infect surrounding tumor cells. The combination of cytopathic effect by oncolytic adenovirus and biological effect of transgene would exert strong antitumor activity. These new types of vectors may provide a potent and safe tool for cancer gene therapy.     

Oncolytic adenoviruses armed with thymidine kinase can be traced by PET imaging and show potent antitumoural effects by ganciclovir dosing

Replication-competent adenoviruses armed with thymidine kinase (TK) combine the concepts of virotherapy and suicide gene therapy. Moreover TK-activity can be detected by noninvasive positron emission-computed tomography (PET) imaging, what could potentially facilitate virus monitoring in vivo. Here, we report the generation of a novel oncolytic adenovirus that incorporates the Tat8-TK gene under the control of the Major Late Promoter in a highly selective backbone thus providing selectivity by targeting the retinoblastoma pathway. The selective oncolytic TK virus, termed ICOVIR5-TK-L, showed reduced potency compared to a non-selective counterpart. However the combination of ICOVIR5-TK-L with ganciclovir (GCV) induced a potent antitumoural effect similar to that of wild type adenovirus in a preclinical model of pancreatic cancer. Although the treatment with GCV provoked a reduction in the viral yield, both in vitro and in vivo, a two-cycle treatment of virus and GCV resulted in an enhanced antitumoral response that correlated with high TK-activity, based on microPET measurements. Thus, TK-expressing oncolytic adenoviruses can be traced by PET imaging providing real time information on the activity of the virus and its antitumoral potency can be optimized by GCV dosing.     

Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific MicroRNA

MicroRNAs have emerged as important players in tissue-specific mammalian gene regulation and have also been exploited in experimental targeting of gene expression. We have constructed a recombinant adenovirus that contains sequences complementary to the liver-specific microRNA 122 (miR122) in the 3′ untranslated region of the E1A gene. In Huh7 cells, which resemble normal hepatocytes in expressing high levels of miR122, this feature resulted in strongly reduced levels of E1A mRNA and protein. This property allowed us to generate a novel recombinant adenovirus that was severely attenuated in cells of hepatic origin but replicated normally in other cells. This strategy may be useful in circumventing liver toxicity associated with the systemic delivery of oncolytic adenoviruses. These data provide the first example of exploiting differential microRNA expression patterns to alter the natural tropism of a DNA virus. In addition, these results suggest that other microRNAs expressed in a tissue- or transformation-specific manner may also be used for the targeting of adenoviral replication and that the same principle may be applied to other viruses that have shown promise as oncolytic or gene delivery platforms.     

A truncated minimal-E1a gene with potency to support adenoviral replication mediates antitumor activity by down-regulating Neu expression and preserving Rb function

Oncolytic adenovirus is capable of infecting, replicating in and lysing cancer cells. In adenovirus infection and replication, the wild type E1a gene (wE1a) mediates various genetic events to facilitate viral replication and exert antitumor effect. To enhance its antitumor efficacy and optimize its safety, we manipulated the wE1a gene and designed a 720-bp truncated minimal-E1a (mE1a) by deletions and mutations of amino acid residues. The mE1a gene was incorporated in an adenovirus under the control of hTERT promoter, giving the vector AdDC315-mE1a. A variety of cancer cell lines infected with the virus expressed the mE1a protein and showed considerable down-regulation in Neu protein expression as compared to normal cell lines. mE1a also had a lower binding affinity to the Rb protein, preserving the Rb tumor suppressive function. The mE1a expression allowed efficient adenovirus replication with high and stable replication ratios in cancer cells (about 125- to 8500-fold higher at 48 h and 180- to 10,900-fold higher at 96 h post-infection). Further, the mE1a-supported oncolytic adenovirus induced higher cancer cell apoptosis, stronger cell cycle arrest and more effective antitumor efficacy in hepatocarcinoma xenografts in nude mice. In conclusion, the truncated minimal mE1a can act as a tumor inhibitor gene, and may be used to construct oncolytic adenovirus vectors for use in gene therapy of a variety of cancers.     

Dichloroacetate (DCA) enhances tumor cell death in combination with oncolytic adenovirus armed with MDA-7/IL-24

Dichloroacetate (DCA) is a metabolic modulator for the treatment of lactic acidosis and inherited mitochondrial diseases. A recent study showed that DCA treatment could induce apoptosis in many kinds of tumor cell lines via mitochondrial apoptotic pathway while sparing normal cells. ONYX-015 (dl 1520) is one of the oncolytic adenoviruses developed by the deletion of E1B-55kD gene of type 5 adenoviral DNA, and it replicates efficiently and selectively in tumor cells. ZD55-IL-24, an E1B-55kD deleted oncolytic adenovirus carrying interleukin-24 (IL-24, also called melanoma differentiation associated gene-7), had showed potent antitumor efficacy in a variety of tumor cells and exerted no apparent toxicity on normal cells. Given both the good therapeutic effect and low toxicity of these agents, here we investigated whether DCA in combination with ZD55-IL-24 or ONYX-015 could have more efficient antitumor activity in vitro experiments. Therefore, we tested the cytotoxicity of combination therapy in normal hepatic cells L-02 and QSG-7701 using the MTT assay. Our results showed that DCA combined with ONYX-015 or ZD55-IL-24 exhibited more potent antitumor activity than DCA or virus alone, and the combination treatment did not have superimposed toxicities in normal cells. Thus, a novel combination therapy associating oncolytic adenoviruses with relatively low toxic drug without severe side effects was proposed.     

Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.     

Productive replication of human adenoviruses in mouse epidermal cells

In contrast to most cells of mouse origin, cell lines derived from mouse epidermis are permissive for replication of human adenovirus type 5. The extent of epidermal cell differentiation correlated with the level of E1A expression and virus replication. Mouse epidermal cells may provide useful models for cancer therapy using replication-competent human adenoviruses.     

Productive replication of human adenovirus type 5 in canine cells

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.     

MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.     

Targeted and armed oncolytic adenovirus via chemoselective modification

Oncolytic adenoviruses (Ads) are an emerging alternative therapy for cancer; however, clinical trial have not yet demonstrated sufficient efficacy. When oncolytic Ads are used in combination with taxoids a synergistic increase in both cytotoxicity and viral replication is observed. In order to generate a next generation oncolytic adenovirus, virion were physically conjugated to a highly potent taxoid, SB-T-1214, and a folate targeting motif. Conjugation was enabled via the metabolic incorporation of non-canonical monosaccharides (O-GlcNAz) and amino acids (homopropargylglycine), which served as sites for chemoselective modification.     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Oncolytic adenovirus: A tool for cancer therapy in combination with other therapeutic approaches

Cancer therapy using oncolytic viruses is an emerging area, in which viruses are engineered to selectively propagate in tumor tissues without affecting healthy cells. Because of the advantages that adenoviruses (Ads) have over other viruses, they are more considered. To achieve tumor selectivity, two main modifications on Ads genome have been applied: small deletions and insertion of tissue- or tumor-specific promoters. Despite oncolytic adenoviruses ability in tumor cell lysis and immune responses stimulation, to further increase their antitumor effects, genomic modifications have been carried out including insertion of checkpoint inhibitors and antigenic or immunostimulatory molecules into the adenovirus genome and combination with dendritic cells and chemotherapeutic agents. This study reviews oncolytic adenoviruses structures, their antitumor efficacy in combination with other therapeutic strategies, and finally challenges around this treatment approach.     

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.