Replication-competent Adenovirus Research Articles

Oncolytic adenovirus-mediated short hairpin RNA targeting MYCN gene induces apoptosis by upregulating RKIP in neuroblastoma.

The amplification of MYCN is a typical characteristic of aggressive neuroblastomas, whereas acquired mutations of p53 lead to refractory and relapsed cases. We had previously examined the applicability of the replication-competent oncolytic adenovirus, ZD55-shMYCN, to deliver a short hairpin RNA targeting MYCN gene for p53-null and MYCN-amplified neuroblastoma cell line LA1-55N. Our data have shown that ZD55-shMYCN has an additive tumor growth inhibitory response through shRNA-mediated MYCN knockdown and ZD55-mediated cancer cell lysis. In this regard, ZD55-shMYCN can downregulate MYCN and perform anticancer effects, thereby acquiring significance in the administration of MYCN-amplified and p53-null neuroblastomas. Hence, we further investigated the anticancer properties of ZD55-shMYCN in neuroblastomas. Our data showed that ZD55-shMYCN induced G2/M arrest via decreasing the levels of cyclin D1 and cyclin B1 irrespective of p53 status. ZD55-shMYCN effectively induced apoptosis in neuroblastomas through activation of caspase-3 and enhancing PARP cleavage. Furthermore, ZD55-shMYCN could downregulate phosphoinositide 3-kinase and pAkt and upregulate RKIP levels. Similarly, pro-apoptosis was revealed by the histopathologic examination of paraffin-embedded section of resected tumors of mice xenograft. In vitro and in vivo studies, we elucidate the apoptosis properties and mechanisms of action of ZD55-shMYCN, which provide a promising approach for further clinical development.

A Combinatory Strategy for Detection of Live CTCs Using Microfiltration and a New Telomerase-Selective Adenovirus.

Circulating tumor cells (CTC) have become an important biomarker for early cancer diagnosis, prognosis, and treatment monitoring. Recently, a replication-competent recombinant adenovirus driven by a human telomerase gene (hTERT) promoter was shown to detect live CTCs in blood samples of patients with cancer. Here, we report a new class of adenoviruses containing regulatory elements that repress the hTERT gene in normal cells. Compared with the virus with only the hTERT core promoter, the new viruses showed better selectivity for replication in cancer cells than in normal cells. In particular, Ad5GTSe, containing three extra copies of a repressor element, displayed a superior tropism for cancer cells among leukocytes and was thus selected for CTC detection in blood samples. To further improve the efficiency and specificity of CTC identification, we tested a combinatory strategy of microfiltration enrichment using flexible micro spring arrays and Ad5GTSe imaging. Our experiments showed that this method efficiently detected both cancer cells spiked into healthy blood and potential CTCs in blood samples of patients with breast and pancreatic cancer, demonstrating its potential as a highly sensitive and reliable system for detection and capture of CTCs of different tumor types.

Oncolytic adenovirus expressing interferon alpha in a syngeneic Syrian hamster model for the treatment of pancreatic cancer

The addition of interferon (IFN) alpha to adjuvant chemoradiotherapy regimens resulted in remarkable improvements in survival for pancreatic cancer patients. However, systemic toxicities and insufficient levels of IFN at the tumor sites have limited its widespread adoption in treatment schemes. We have previously developed an IFN-expressing conditionally replicative oncolytic adenovirus and demonstrated its therapeutic effects both in vitro and in vivo. Here, the same vectors were tested in a syngeneic and immunocompetent Syrian hamster model to better understand the roles of adenoviral replication and of the pleiotropic effects of IFN on pancreatic tumor growth suppression. Oncolytic adenoviruses expressing human or hamster IFN were designed and generated. Viral vectors were tested in vitro to determine qualitative and quantitative cell viability, cyclooxygenase 2 (Cox2) promoter activity, and IFN production. For the in vivo studies, subcutaneous hamster pancreatic cancer tumors were treated with 1 intratumoral dose of virus. Similarly, 1 intraperitoneal dose of virus was used to prolong survival in a carcinomatosis model. All cell lines tested demonstrated Cox2 promoter activity. The oncolytic potential of a replication competent adenovirus expressing the IFN cytokine was clearly demonstrated. These viruses resulted in significant tumor growth suppression and survival increases compared with controls in a hamster model. The profound therapeutic potential of an IFN-expressing oncolytic adenovirus for the treatment of pancreatic cancer was demonstrated in a syngeneic Syrian hamster model. These results strongly suggest the potential application of our viruses as part of combination regimens with other therapeutics.

Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells.

Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3'UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3'UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses.

A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.

Abstract B50: A novel immunocompetent murine model for replicating oncolytic adenoviral therapy

Abstract Oncolytic adenoviruses are able to replicate selectively in cancerous but not normal human cells and are under investigation as potential cancer therapeutics. Unfortunately, mice are poor model systems for therapy with replication competent adenoviruses because murine cells are generally unable to produce infectious viral progeny. In order to model oncolytic adenovirus treatment, researchers have used human tumor xenografts that support viral replication in immunodeficient mice. Because xenografts require an immunodeficient host, xenograft model systems do not reflect a host's adaptive immune response against the virus and cannot model the potential for an immunostimulatory virus to induce an anti-tumor immune response. Using the tumor-selective oncolytic adenovirus TAV-255 previously developed in our laboratory, we find that the murine lung adenocarcinoma cell line LKR-13 supports adenoviral infection and generates infectious viral progeny. Western blotting showed that LKR-13 cells express the coxsackie and adenovirus receptor (CAR) to allow viral infection, and that infected cells express the viral protein E1A. MTT assays, crystal violet staining, and clonogenic assays showed that TAV-255 kills LKR-13 cells in a dose-dependent and time-dependent manner with similar potency and latency of infection as a panel of human cancer cell lines, with more than 50% cell death of murine LKR-13 cells and various human cancer cells (T1, 882 and A549) within 6 days of infection at a multiplicity of infection of 10. It produces infectious progeny from LKR-13 cells at titres similar to other human cancer cell lines. Other tested murine cancer cell lines do not share these features; for example, the CT-26 colon cancer cell line does not express CAR and is resistant TAV-255 infection, while the murine sarcoma cell line F244 is readily infected by adenovirus and produces the viral protein E1A but does not produce infectious progeny. Based on these in-vitro findings, we investigated LKR-13 cells as an in-vivo model system for replicating oncolytic adenoviruses. The LKR-13 cell line is derived from 129/Sv mice, and using fully immunocompetent 129/s4 mice, subcutaneous injection of 5x106 LKR-13 cells led to tumor formation in all injected mice. Intratumoral injection of 3x107 plaque forming units of TAV-255 in established tumors (greater than 200 mm3) caused a significant reduction in tumor growth: Eight days after treatment, tumors in mice treated with TAV-255 were 339 mm3 +/- 26 mm3 (n=3), and tumors in mice treated with PBS were 578 mm3 +/- 49 mm3 (n=5) (p=0.013). This model system represents the first fully immunocompetent mouse model for cancer treatment with replicating oncolytic adenoviruses, and will be useful to study the therapeutic effect of oncolytic adenoviruses in general and particularly immunostimulatory viruses designed to evoke an anti-tumor immune response. Citation Format: Lingzhi Zhang, Farah Hedjran, Christopher Larson, Min Yan, Tony Reid. A novel immunocompetent murine model for replicating oncolytic adenoviral therapy. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B50.

Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions. Furthermore, activation of the cGAS cascade occurred in a cell-specific manner. Activation of the cGAS/STING response did not impact viral replication, and viral immune evasion strategies did not target the cGAS/STING/TBK1/IRF3 cascade. These studies provide novel insight into the early innate recognition response to adenovirus.

Abstract 725: Combination therapy of telomerase-specific oncolytic adenovirus and zoledronic acid in human osteosarcoma cells

Abstract Osteosarcoma is one of the primary malignant bone tumors that often occur around the ages of 15 to 20. Despite advances in combined chemotherapy and surgical resection, 30% of osteosarcoma patients still die from tumor or metastasis. Therefore, new treatment strategy is being required. We previously reported that telomerase-specific, replication-competent oncolytic adenovirus (Telomelysin, OBP-301), showed the in vitro and in vivo antitumor effects in human bone and soft tissue sarcoma cells; however OBP-301 could not efficiently prevent bone destruction in an orthotopic xenograft tumor model. Recently, third-generation bisphosphonates, zoledronic acid (ZOL), is widely used to inhibit bone destruction in metastatic bone tumors in the clinical settings. Moreover, ZOL has been shown to induce the antitumor effect synergistically in combination with chemotherapeutic agents in human sarcoma cells. In this study, we investigated the in vitro and in vivo antitumor effects of combination therapy with ZOL and OBP-301 in human osteosarcoma cells. Four human osteosarcoma cell lines, HOS, SaOS-2, U2OS and MNNG/HOS were used. We used XTT assay to examine the antitumor effect of ZOL and OBP-301 individually and combinatory on days 2,3,5. ZOL was treated at concentration of 0 to 10µM, and OBP-301 was infected at multiplicity of infections (MOI) of 0 to 100 plaque forming units (PFU)/cell. Combination index was calculated with the CalcuSyn software (BioSoft) and combined antitumor effect between ZOL and OBP-301 was analyzed. XTT assay revealed that combination treatment of ZOL and OBP-301 showed synergistic and additive effects. To analyze the molecular mechanism in the synergistic effect of ZOL and OBP-301, western blot analysis for apoptosis (PARP) and autophagy (LC3, p62) was performed. Treatment with ZOL induced apoptosis, which was confirmed by the cleavage of PARP, and OBP-301infection induced both apoptosis and autophagy, which is confirmed by conversion of LC3- I to LC3- II and p62 downregulation. Combination treatment showed increased apoptotic cell death in all human osteosarcoma cells. Moreover, in vivo antitumor effect of combination therapy was investigated in an orthotopic xenograft tumor model, in which MNNG/HOS cells (2×106 cells per site) were inoculated into the left tibias of female athymic nude mice. ZOL and OBP-301 were injected subcutaneously and intratumorally, respectively, for three times every week. Tumor volume was measured once a week and bone destruction status was analyzed using 3D-CT imaging system at final assessment. Combination treatment showed increased antitumor effect and efficiently prevented bone destruction compared to monotherapy in an orthotopic xenograft MNNG/HOS tumor model. These results suggest that combination treatment of ZOL and OBP-301 is a promising antitumor strategy for improvement of clinical outcome in patients with osteosarcomas. Citation Format: Yasuaki Yamakawa, Joe Hasei, Hiroshi Tazawa, Toshinori Omori, Shuhei Osaki, Tsuyoshi Sasaki, Aki Yoshida, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Combination therapy of telomerase-specific oncolytic adenovirus and zoledronic acid in human osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2014-725

Abstract 342: Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation

Abstract Osteosarcoma is the most common malignant primary bone tumor. Despite advances in multi-agent chemotherapy, poor response to chemotherapy is one of the critical prognostic factors in osteosarcoma. Therefore, enhancement of chemosensitivity is one approach to improve the survival of osteosarcoma patients. We recently developed a telomerase-specific replication-competent oncolytic adenovirus, Telomelysin (OBP-301). A Phase I clinical trial in the U.S. showed the safety of OBP-301 in advanced cancer patients, and a Phase I/II clinical trial for malignant thoracic tumors is currently undergoing. We recently confirmed the antitumor effect of OBP-301 monotherapy in human osteosarcoma cells. In this study, we investigated the chemosensitizing effect of OBP-301 in human osteosarcoma cells and the molecular mechanism in the OBP-301-mediated enhancement of cell death. We used four human osteosarcoma cell lines, HOS, MNNG/HOS, 143B and SaOS-2. OBP-301 is an attenuated adenovirus, in which the hTERT promoter drives the expression of E1 gene, and causes tumor-selective lysis in a variety of human malignant tumor cells with telomerase activity. OBP-301 infection enhanced the cytotoxic effect of chemotherapeutic agents, cisplatin and doxorubicin, that are commonly used for the treatment of osteosarcomas. The calculation of combination index revealed the synergistic effects in all human osteosarcoma cell lines. Combination of OBP-301 increased apoptosis in the chemotherapeutic agents-treated cells. To clarify the molecular mechanism for the chemosensitizing effect of OBP-301, the expression of Bcl-2 family proteins, which are critical regulators of apoptosis, were investigated. In OBP-301-infected MNNG/HOS and SaOS-2 cells, the expression level of Mcl-1 was markedly decreased by OBP-301 infection. Downregulation of Mcl-1 expression by siRNA enhanced the chemotherapeutic agents-induced apoptosis. Moreover, combination therapy of chemotherapeutic agents with OBP-301 significantly suppressed tumor growth in a subcutaneous xenograft tumor model compared to monotherapy with chemotherapeutic agents or OBP-301. Anti-apoptotic Mcl-1 protein has been shown to be frequently overexpressed in human sarcomas. Overexpression of anti-apoptotic proteins is an important factor to prevent chemotherapy-induced apoptosis in cancer. We demonstrated the involvement of Mcl-1 suppression in the chemosensitizing effect of OBP-301 in human osteosarcoma cells. These results suggest that Mcl-1 is a critical target for improvement of the chemosensitivity in human osteosarcoma cells and the combination of chemotherapy with OBP-301 may be a novel therapeutic strategy for osteosarcoma patients. Citation Format: Shuhei Osaki, Toshinori Omori, Hiroshi Tazawa, Joe Hasei, Yasuaki Yamakawa, Tsuyoshi Sasaki, Toshiyuki Kunisada, Yasuo Urata, Toshifumi Ozaki, Toshiyoshi Fujiwara. Telomerase-dependent oncolytic adenovirus sensitizes human osteosarcoma cells to chemotherapy through Mcl-1 downregulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2014-342

Comparative analysis of SIV-specific cellular immune responses induced by different vaccine platforms in rhesus macaques

To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.

A Detection System for Circulating Tumor Cells (Ctcs) Using Gfp Expressing Telomerase-Specific Replication-Competent Adenovirus in Bone and Soft Tissue Sarcoma

ABSTRACT Aim: The concept of circulating tumor cell (CTC) is of great interest recently in the field of cancer treatment. CTCs defined as cancer cells of solid tumor origin found in the peripheral blood, and have been detected in the patients with advanced cancer. However, CTCs have also detected in patients with localized cancers, indicating the risk of progression to metastatic disease. Although many papers on CTCs have been published, there is no report on CTCs detected in patients with bone and soft tissue tumors. We analyzed CTCs in the peripheral blood of patients with sarcoma. Methods: 7.5ml of blood was collected from the patients, and CTCs were assessed using TelomeScan (OBP-401), which GFP-expressing attenuated adenovirus-5 vector, in which the hTERT promoter regulates viral replication. A total of 22 patients with bone (6 osteosarcoma) and soft tissue sarcoma (4 myxofibrosarcoma, 4 liposarcoma, 3 DFSP, 2 synovial sarcoma, 3 others) patients were enrolled. GFP-expressing cells were labeled with anti-CD45 (a marker for blood cell), anti-vimentin antibody (a marker for mesenchymal cell) and DAPI stain (a marker for cell nucleus) 24 hours after the virus infection to ensure that cells labeled with GFP were indeed sarcoma cells. Results: CTCs were detected in 11 of 22 sarcoma patients (4 osteosarcoma, 3 myxofibrosarcoma, 2 DFSP, 2 others). Average number of CTCs was 3.5 cells per 7.5ml. 3 patients with positive CTC showed negative CTC after sarcoma resection, and 4 patients with positive CTC showed negative CTC after chemotherapy. 5 of 22 patients developed lung metastasis, 4 of those showed positive CTC at diagnosis. Conclusions: CTCs were detected in 50% of patients with bone and soft tissue sarcoma in our series. CTC detection using TelomeScan has been reported only in patients with cancer. We identified that the detection system using TelomeScan can be a new approach to visualize live human CTCs from bone and soft tissue sarcoma, and that CTC can be also a biomarker for bone and soft tissue sarcoma. Disclosure: All authors have declared no conflicts of interest.

MiR-148a- and miR-216a-regulated Oncolytic Adenoviruses Targeting Pancreatic Tumors Attenuate Tissue Damage Without Perturbation of miRNA Activity

Oncolytic virotherapy shows promise for pancreatic ductal adenocarcinoma (PDAC) treatment, but there is the need to minimize associated-toxicities. In the current work, we engineered artificial target sites recognized by miR-216a and/or miR-148a to provide pancreatic tumor-selectivity to replication-competent adenoviruses (Ad-miRTs) and improve their safety profile. Expression analysis in PDAC patients identified miR-148a and miR-216a downregulated in resectable (FC(miR-148a) = 0.044, P < 0.05; FC(miR-216a) = 0.017, P < 0.05), locally advanced (FC(miR-148a) = 0.038, P < 0.001; FC(miR-216a) = 0.001, P < 0.001) and metastatic tumors (FC(miR-148a) = 0.041, P < 0.01; FC(miR-216a) = 0.002, P < 0.001). In mouse tissues, miR-216a was highly specific of the exocrine pancreas whereas miR-148a was abundant in the exocrine pancreas, Langerhans islets, and the liver. In line with the miRNA content and the miRNA target site design, we show E1A gene expression and viral propagation efficiently controlled in Ad-miRT-infected cells. Consequently, Ad-miRT-infected mice presented reduced pancreatic and liver damage without perturbation of the endogenous miRNAs and their targets. Interestingly, the 8-miR148aT design showed repressing activity by all miR-148/152 family members with significant detargeting effects in the pancreas and liver. Ad-miRTs preserved their oncolytic activity and triggered strong antitumoral responses. This study provides preclinical evidences of miR-148a and miR-216a target site insertions to confer adenoviral selectivity and proposes 8-miR148aT as an optimal detargeting strategy for genetically-engineered therapies against PDAC.

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications.

A genetic fiber modification to achieve matrix-metalloprotease-activated infectivity of oncolytic adenovirus.

Selective tumor targeting of oncolytic adenovirus at the level of cell entry remains a major challenge to improve efficacy and safety. Matrix metalloproteases (MMPs) are overexpressed in a variety of tumors and in particular in pancreatic cancer. In the current work, we have exploited the expression of MMPs together with the penetration capabilities of a TAT-like peptide to engineer tumor selective adenoviruses. We have generated adenoviruses containing CAR-binding ablated fibers further modified with a C-terminus TAT-like peptide linked to a blocking domain by an MMP-cleavable sequence. This linker resulted in a MMP-dependent cell transduction of the reporter MMP-activatable virus AdTATMMP and in efficient transduction of neoplastic cells and cancer-associated fibroblasts. Intravenous and intraductal administration of AdTATMMP into mice showed very low AdTATMMP activity in the normal pancreas, whereas increased transduction was observed in pancreatic tumors of transgenic Ela-myc mice. Intraductal administration of AdTATMMP into mice bearing orthotopic tumors led to a 25-fold increase in tumor targeting compared to the wild type fiber control. A replication competent adenovirus, Ad(RC)MMP, with the MMP-activatable fiber showed oncolytic efficacy and increased antitumor activity compared to Adwt in a pancreatic orthotopic model. Reduced local and distant metastases were observed in Ad(RC)MMP treated-mice. Moreover, no signs of pancreatic toxicity were detected. We conclude that MMP-activatable adenovirus may be beneficial for pancreatic cancer treatment.

Dissecting the roles of E1A and E1B in adenoviral replication and RCAd-enhanced RDAd transduction efficacy on tumor cells

Oncolytic viruses have recently received widespread attention for their potential in innovative cancer therapy. Many telomerase promoter-regulated oncolytic adenoviral vectors retain E1A and E1B. However, the functions of E1A and E1B proteins in the oncolytic role of replication-competent adenovirus (RCAd) and RCAd enhanced transduction of replication defective adenoviruses (RDAd) have not been addressed well. In this study, we constructed viruses expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa. We then tested their roles in oncolysis and replication of RCAd as well as their roles in RCAd enhanced transfection rate and transgene expression of RDAd in various cancer cells in vitro and in xenografted human NCI-H460 tumors in nude mice. We demonstrated that RCAds expressing E1A alone and plus E1B-19 kDa exhibited an obvious ability in replication and oncolytic effects as well as enhanced RDAd replication and transgene expression, with the former showed more effective oncolysis, while the latter exhibited superior viral replication and transgene promotion activity. However, RCAd expressing both E1A and E1B-19 kDa/55 kDa was clearly worst in all these abilities. The effects of E1A and E1B observed through using RCAd were further validated by using plasmids expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa proteins. Our study provided evidence that E1A was essential for inducing replication and oncolytic effects of RCAd as well as RCAd enhanced RDAd transduction, and expression of E1B-19 kDa other than E1B-55 kDa could promote these effects. E1B-55 kDa is not necessary for the oncolytic effects of adenoviruses and somehow inhibits RCAd-mediated RDAd replication and transgene expression.

IIIa deleted adenovirus as a single-cycle genome replicating vector

Replication competent adenovirus (RC-Ad) vectors mediate robust transgene expression by virtue of amplifying transgenes by replication but also put patients at a risk of frank adenovirus infection. In contrast, E1-deleted replication defective Ad (RD-Ad) vectors are safer but produce substantially less transgene product. To generate a robust, but safer adenoviral vector, we created a “single cycle” adenovirus (SC-Ad) vector that replicates its genome and transgene, but that does not cause adenovirus infections by deleting the capsid cement protein IIIa in low seroprevalence adenovirus serotype 6. Ad6-ΔIIIa can be produced in IIIa-expressing cell lines. In normal cells, Ad6-ΔIIIa replicates its genome and transgene but fails to package its DNA or form mature virus. SC-Ad and RC-Ad expressed transgenes hundreds of times higher than RD-Ad in human and mouse cells in vitro and in vivo in mice. These data suggest that SC-Ads may be safer amplifying vectors for vaccine and therapeutic applications.

Development of replication-competent adenovirus for bladder cancer by controlling adenovirus E1a and E4 gene expression with the survivin promoter.

Survivin is a member of the inhibitors of apoptosis protein family. Here, we examined survivin expression and confirmed abundant survivin expression in bladder cancer cells. This expression pattern indicated that the transcriptional regulatory elements that control survivin expression could be utilized to discriminate cancer from normal cells. We therefore generated a novel adenovirus termed Ad5/35E1apsurvivinE4 with the following characteristics: 1) E1A and E4 protein expression was dependent on survivin promoter activity; 2) the green fluorescence protein gene was inserted into the genome under the control of the CMV promoter; 3) most of the E3 sequences were deleted, but the construct was still capable of expressing the adenovirus death protein with potent cytotoxic effects; and 4) the fiber knob was from serotype 35 adenovirus. As expected from the abundant survivin expression observed in bladder cancer cells, Ad5/35E1apsurvivinE4 replicated better in cancer cells than in normal cells by a factor of 106 to 102. Likewise, Ad5/35E1apsurvivinE4 exerted greater cytotoxic effects on all bladder cancer cell lines tested. Importantly, Ad5/35E1apsurvivinE4 inhibited the growth of Ku7-Luc orthotopic xenografts in nude mice. Taken together, Ad5/35E1apsurvivinE4 indicates that the survivin promoter may be utilized for the development of a replication-competent adenovirus to target bladder cancers.

Replication-competent adenovirus expressing TRAIL synergistically potentiates the antitumor effect of gemcitabine in bladder cancer cells

Replication-competent adenovirus armed with therapeutic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been shown to sensitize cancer cells to chemotherapy and radiotherapy. However, the synergistic antitumor effect of replication-competent adenovirus expressing TRAIL and the cytotoxic chemotherapy in bladder cancer remains to be determined. Bladder cancer T24 cells or mouse tumor xenografts were infected with replication-competent adenovirus armed with human TRAIL (ZD55-TRAIL) alone or in combination with gemcitabine. The mRNA and protein levels of TRAIL were determined by "Reverse transcription polymerase chain reaction" and Western blotting, respectively. Cell viability was tested by CCK8 assay. Tumor growth in the mice was monitored every week by measuring tumor size. Cell apoptosis was detected by Annexin V-FITC staining and TUNEL assay. We found that adenovirus ZD55-TRAIL efficiently replicated both in cultured bladder cancer T24 cells and T24 mouse tumor xenograft as demonstrated by the overexpression of TRAIL and E1A. Gemcitabine did not affect the expression of TRAIL. In cultured T24 cells, ZD55-TRAIL enhanced the growth inhibitory effects of gemcitabine, accompanied by increased apoptosis. Similarly, ZD55-TRAIL synergistically enhanced the antitumor effect and induction of apoptosis following gemcitabine treatment in mouse T24 xenografts. In conclusion, replicative adenovirus armed with TRAIL synergistically potentiates the antitumor effect of gemcitabine in human bladder cancer. Our study provides the basis for the development of ZD55-TRAIL in combination with conventional chemotherapy for the treatment of bladder cancer.

Chemoprevention Gene Therapy (CGT) of Pancreatic Cancer Using Perillyl Alcohol and a Novel Chimeric Serotype Cancer Terminator Virus

Conditionally replication competent adenoviruses (Ads) that selectively replicate in cancer cells and simultaneously express a therapeutic cytokine, such as melanoma differentiation associated gene- 7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7), hold potential for treating human cancers. To enhance the efficacy of the CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiber bipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a Coxsackie Adenovirus receptor (CAR) independent manner, while retaining high infectivity in cancer cells containing high CAR. Although mda-7/IL-24 displays broad-spectrum anticancer properties, pancreatic ductal adenocarcinoma (PDAC) cells display an intrinsic resistance to mda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translational block. However, using a chemoprevention gene therapy (CGT) approach with perillyl alcohol (POH) and a replication incompetent Ad to deliver mda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNA into protein resulting in pancreatic cancer cell death in vitro and in vivo in nude mice containing human PDAC xenografts. This combination synergistically induces mda-7/IL-24-mediated cancer-specific apoptosis by inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression and inducing an endoplasmic reticulum (ER) stress response through induction of BiP/GRP-78, which is most evident in chimeric-modified non-replicating Ad.5/3- mda-7- and CTV-M7-infected PDAC cells. Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizes therapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 or Bcl-xL to mda-7/IL-24-mediated apoptosis. Ad.5/3-CTV-M7 plus POH also exerts a significant antitumor 'bystander' effect in vivo suppressing both primary and distant site tumor growth, confirming therapeutic utility of Ad.5/3-CTV-M7 plus POH in PDAC treatment, where all other current treatment strategies in clinical settings show minimal efficacy.

Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy

Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used for gene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essential viral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used for gene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer gene therapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through the natural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competent adenovirus vectors are safe and have therapeutic activity.