Increased antitumor capability of fiber-modified adenoviral vector armed with TRAIL against bladder cancers

Adenoviral vectors are widely used for cancer therapy and show a tumor-suppressing effect. However, bladder cancers are found to be resistant against infection of Ad5-derived adenoviral vector, limiting the application of the existing strategy of gene therapy. Therefore, efforts to develop novel types of adenoviral vector aimed for improving the viral infection and enhancing expression level of tumor-inhibiting transgene is urgently required. We constructed a 5/35 fiber-modified E1A-deleted adenoviral vector armed with TRAIL gene. Its ability to express this gene for inhibition of bladder cancer cell growth was investigated in our work. The results showed that this modification in fiber region facilitates adenoviral infection to bladder cancer, perhaps due to high expression of CD46 on target cell surface. Subsequently, we found an enhanced expression level of TRAIL mediated by 5/35 fiber-modified adenoviral vectors in bladder cancer cells, leading to an increased tumor-inhibiting capability of 5/35 adenoviral vector against bladder cancer cells. Consistently, growth of xenograft tumors in mice was also effectively inhibited by 5/35 fiber-modified vector-mediated gene therapy strategy. The 5/35 fiber-modified adenoviral vector-based gene transfer shows an improved efficacy against bladder cancers. The application of this novel gene therapy vector may benefit the patients in clinical bladder cancer treatment.     

A new oncolytic adenoviral vector carrying dual tumour suppressor genes shows potent anti-tumour effect

Cancer Targeting Gene-Viro-Therapy (CTGVT) is a promising cancer therapeutical strategy that strengthens the anti-tumour effect of oncolytic virus by expressing inserted foreign anti-tumour genes. In this work, we constructed a novel adenoviral vector controlled by the tumour-specific survivin promoter on the basis of the ZD55 vector, which is an E1B55KD gene deleted vector we previously constructed. Compared with the original ZD55 vector, this new adenoviral vector (ZD55SP/E1A) showed much better ability of replication and reporter gene expression. We then combined anti-tumour gene interleukine-24 (IL-24) with an RNA polymerase III-dependent U6 promoter driving short hairpin RNA (shRNA) that targets M-phase phosphoprotein 1 (MPHOSPH1, a newly identified oncogene) by inserting the IL-24 and the shRNA of MPHOSPH1 (shMPP1) expression cassettes into the new ZD55SP/E1A vector. Our results demonstrated excellent anti-tumour effect of ZD55SP/E1A-IL-24-shMPP1 in vitro on multiple cancer cell lines such as lung cancer, liver cancer and ovarian caner. At high multiplicity-of-infection (MOI), ZD55SP/E1A-IL-24-shMPP1 triggered post-mitotic apoptosis in cancer cells by inducing prolonged mitotic arrest; while at low MOI, senescence was induced. More importantly, ZD55SP/E1A-IL-24-shMPP1 also showed excellent anti-tumour effects in vivo on SW620 xenograft nude mice. In conclusion, our strategy of constructing an IL-24 and shMPP1 dual gene expressing oncolytic adenoviral vector, which is regulated by the survivin promoter and E1B55KD deletion, could be a promising method of cancer gene therapy.     

Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL

Chemoresistance and side effects are considered as the major obstacles in cisplatin-based chemotherapy of various human malignant tumors. Conjugation with cancer-specific apoptotic stimuli TRAIL or typical viro-agent ONYX-015 has been extensively investigated to enhance the antitumor activity of cisplatin. In this study, we presented a novel chemo-gene-virotherapeutic strategy to further improve the toxic effects in cancer cells and reduce the damage in normal cells. Here, an oncolytic adenoviral vector (ZD55), with a deletion of E1B 55-kDa gene, was employed to express the therapeutic TRAIL gene by constructing a recombinant virus ZD55-TRAIL. Exogenous gene delivery efficacy was determined by both in vitro and in vivo experiments and enhanced cytotoxicity of combined treatment of ZD55-TRAIL with cisplatin was evaluated in several cancer cell lines. Moreover, negative effects on normal cells have been tested in both L-02 and MRC-5 cell lines by MTT assay and apoptotic cell staining. According to our observation, combination of ZD55-TRAIL with cisplatin exhibited an apparent synergistic cytotoxicity in cancer cells, yet significantly abolished the negative toxicity in normal cells by reducing the dosage. Thus, a novel chemo-gene-virotherapeutic strategy for cancer therapy was proposed.     

Adenoviral-mediated transfer of the TNF-related apoptosis-inducing ligand/Apo-2 ligand gene induces tumor cell apoptosis

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily of cytokines that induces apoptosis in a variety of cancer cells. The results presented in this study demonstrate that introduction of the human TRAIL gene into TRAIL-sensitive tumor cells using an adenoviral vector leads to the rapid production and expression of TRAIL protein, and subsequent death of the tumor cells. Tumor cell death was mediated by an apoptotic mechanism, as evidenced by the activation of caspase-8, cleavage of poly(ADP-ribose) polymerase, binding of annexin V, and inhibition by caspase inhibitor zVAD-fmk. These results define a novel method of using TRAIL as an antitumor therapeutic, and suggest the potential use for an adenovirus-encoding TRAIL as a method of gene therapy for numerous cancer types in vivo.     

A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight,inducible transgene expression

Background

One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.

Results

We constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.

Conclusions

Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.     

MiRNA regulation of TRAIL expression exerts selective cytotoxicity to prostate carcinoma cells

Prostate carcinoma is the most common cancer for men and among the leading cancer-related causes. Many evidences have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in cancer cells, and thus, is a promising biologic agent for prostate carcinoma therapy. However, TRAIL expression mediated by the current vectors lacks tumor specificity, thereby exerting cytotoxicity to normal cells. To solve this problem, we inserted miRNA response elements (MREs), miR-143 and miR-145, expression levels of which were reduced in prostate carcinoma, as well as that of miR-122, which is specifically expressed in hepatic cells, into adenoviral vectors to control TRAIL expression (Ad-TRAIL-M3). qPCR data confirmed that miR-143, miR-145, and miR-122 levels were all decreased in prostate carcinoma cell lines and prostate cancer samples from patients. Luciferase assays showed that MREs-regulated luciferase expression was potently suppressed in normal cells, but not in prostate cancer cells. Ad-TRAIL-M3, which expresses TRAIL in a MREs-regulated manner, produced high level of TRAIL and suppressed the survival of prostate cancer cells by inducing apoptosis, while Ad-TRAIL-M3 had no TRAIL expression in normal cells and thus exerted no cytotoxicity to them. The studies on PC-3 tumor xenograft in mice further confirmed that Ad-TRAIL-M3 was able to inhibit the growth of tumors and possessed high biosafety. In conclusion, we successfully generated an adenoviral vector that expresses TRAIL in miRNA-regulated mechanism. This miRNA-based gene therapy may be promising for prostate carcinoma treatment.     

Selective TRAIL‐induced cytotoxicity to lung cancer cells mediated by miRNA response elements

Lung cancer is among the most common cancers, and the current therapeutic strategies are still inefficient in most cases. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising biological agent for cancer treatment because of its potent pro‐apoptotic effect on cancer cells. However, TRAIL also induces apoptosis in normal cells and therefore may cause toxicity to normal tissues if clinically applied. To address this issue, we inserted microRNA response elements (MREs) of miR‐133a, miR‐137 and miR‐449a, which are all underexpressed in lung cancer cells, into an adenoviral vector to regulate TRAIL expression. This MRE‐regulated vector (Ad‐TRAIL‐MRE) was able to express TRAIL in a lung‐cancer‐specific fashion. No TRAIL expression was detected in normal cells. Consistently, Ad‐TRAIL‐MRE exerted cytotoxicity to lung cancer cells, rather than normal cells, perhaps via inducing selective apoptosis. The selective TRAIL‐mediated growth‐inhibiting effect was further confirmed in a tumour xenograft model. Also, Ad‐TRAIL‐MRE only resulted in very low hepatotoxicity when applied. Collectively, we generated a novel TRAIL‐expressing adenoviral vector that was regulated by MREs. This strategy permits TRAIL expression in a lung‐cancer‐specific manner and is worth further studying for clinical trials. Copyright © 2014 John Wiley & Sons, Ltd.     

Production of lentiviral vectors by transient expression of minimal packaging genes from recombinant adenoviruses

BACKGROUND: The potential of lentiviral vectors for clinical gene therapy has not yet been evaluated. One of the reasons is the cytotoxicity of lentiviral packaging genes which makes the generation of stable producer cell lines difficult. Therefore, a novel packaging system for lentiviral vectors based on transient expression of packaging genes by recombinant adenoviruses was developed. METHODS: Adenoviral vectors expressing VSV-G, codon-optimized HIV-1 gag-pol, and codon-optimized SIV gag-pol under the control of a tetracycline-regulatable promoter (adenoviral lenti-pack vectors) were constructed and the production levels of this vector system were evaluated. RESULTS: The generated adenoviral lenti-pack vectors could be grown to high titers when transgene expression was suppressed and no evidence for instabilities was obtained. Cells stably transfected with a SIV-based vector construct were converted into lentiviral vector producer cells by infection with the adenoviral lenti-pack vectors. Lentiviral vector titers obtained were as high as vector titers obtained by transient cotransfection experiments. A protocol was developed that allowed preparation of lentiviral vector stocks with undetectable levels of contaminating adenoviral lenti-pack vectors. CONCLUSIONS: The adenoviral lenti-pack vectors described should provide a convenient alternative approach to inducible packaging cell lines for large-scale lentiviral vector production. Transient expression of cytotoxic lentiviral packaging genes by the adenoviral lenti-pack vectors circumvents loss of titers during prolonged culture of packaging cell lines. The design of the adenoviral lenti-pack vectors should reduce the risk of transfer of packaging genes to target cells and at the same time provide flexibility with respect to the lentiviral vector constructs that can be packaged.     

Epidermal growth factor receptor targeting enhances adenoviral vector based suicide gene therapy of osteosarcoma

Background

Despite improvements in the treatment of osteosarcoma (OS) there are still too many patients who cannot benefit from current treatment modalities. Therefore, new therapeutic approaches are warranted. Here we explore the efficacy of targeted adenoviral based suicide gene therapy.

Methods and results

Immunohistochemistry and FACS analysis detected low or absent expression levels of the primary adenovirus receptor CAR on human primary OS and human OS cell lines. These results predict a low infection efficiency and thus a reduced therapeutic effect. Targeting the adenoviruses to another receptor highly expressed on OS could overcome this limitation. We found epidermal growth factor receptor (EGFR) to be widely expressed on primary OS. Immunohistochemistry on primary tumor samples and FACS analysis on primary short‐term cultures and four OS cell lines showed that EGFR was consistently expressed. The recombinant bispecific single‐chain antibody 425‐s11 redirects adenoviral vectors towards the EGFR. Adenovirus transduction experiments in the presence or absence of 425‐s11 showed significantly enhanced gene transfer with the targeted adenoviral vector compared with the native vector (OS cell lines 2.5 to 7.2 times enhanced gene transfer and OS primary short term cultures 1.7 to 10 times enhanced gene transfer). On this basis, targeted suicide gene therapy experiments with AdCMVHSV‐TK in combination with ganciclovir were performed. These experiments demonstrated up to 3.5‐fold enhanced kill of OS cell lines and primary short‐term cultures by the EGFR targeted vector.

Conclusions

Suicide gene therapy with adenovirus targeted towards EGFR may have favorable therapeutic characteristics for future gene therapy applications in OS. Copyright © 2002 John Wiley & Sons, Ltd.

     

Induction of apoptosis by tumor suppressor FHIT via death receptor signaling pathway in human lung cancer cells

FHIT is a novel tumor suppressor gene located at human chromosome 3p14.2. Restoration of wild-type FHIT in 3p14.2-deficient human lung cancer cells inhibits cell growth and induces apoptosis. In this study, we analyzed potential upstream/downstream molecular targets of the FHIT protein and found that FHIT specifically targeted and regulated death receptor (DR) genes in human non-small-cell lung cancer (NSCLC) cells. Exogenous expression of FHIT by a recombinant adenoviral vector (Ad)-mediated gene transfer upregulated expression of DR genes. Treatment with a recombinant TRAIL protein, a DR-specific ligand, in Ad-FHIT-transduced NSCLC cells considerably enhanced FHIT-induced apoptosis, further demonstrating the involvement of DRs in FHIT-induced apoptosis. Moreover, we also found that FHIT targeted downstream of the DR-mediated signaling pathway. FHIT overexpression disrupted mitochondrial membrane integrity and activated multiple pro-apoptotic proteins in NSCLC cell. These results suggest that FHIT induces apoptosis through a sequential activation of DR-mediated pro-apoptotic signaling pathways in human NSCLC cells.     

A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication

Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.     

Specific resistance upon lentiviral TRAIL transfer by intracellular retention of TRAIL receptors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in many transformed cells, suggesting TRAIL as an ideal candidate for cancer gene therapy. A main obstacle in cancer therapy is intrinsic or acquired therapy resistance of malignant cells. To study induction of resistance against TRAIL, we generated lentiviral vectors allowing efficient TRAIL expression and apoptosis induction in a variety of human cancer cell lines. Within days upon TRAIL overexpression, cells became resistant towards TRAIL, but not to CD95 ligation or DNA damage by cisplatin. Cell surface expression of TRAIL receptors 1 and 2 was completely abrogated in resistant cells due to intracellular retention of the receptors by TRAIL. SiRNA directed against TRAIL resensitized the resistant cells by restoring cell surface expression of TRAIL receptors. These findings represent a novel resistance mechanism towards TRAIL, specifically caused by TRAIL overexpression, and question the use of TRAIL expression in tumor-cell targeting gene therapy.     

Survivin silencing and TRAIL expression using oncolytic adenovirus increase anti-tumorigenic activity in gemcitabine-resistant pancreatic cancer cells

In this study, we demonstrated that survivin downregulation with TRAIL expression greatly enhanced the cytotoxic death of pancreatic cancer cells after gemcitabine treatment. Using real-time RT-PCR, we analyzed five survivin shRNAs to identify the best target sequence for suppression of human survivin, with the goal of treating gemcitabine-resistant pancreatic cancer cells. Survivin shRNA 5, corresponding to target 5, showed the greatest reduction in survivin mRNA levels. Furthermore, combined treatment with survivin shRNA-expressing adenovirus with gemcitabine plus TRAIL decreased uncleaved PARP and increased consequent PARP cleavage, which was correlated with the greatest levels of survivin downregulation and cell death. These results indicate that survivin functions as a common mediator of gemcitabine- and TRAIL-induced cell death. Using a nude mouse model implanted with MiaPaCa-2 pancreatic cancer cells, we observed tumor regression induced by an oncolytic adenovirus expressing survivin shRNA and TRAIL plus gemcitabine. Together, our findings provide a strong rationale for treating pancreatic cancer patients with both gemcitabine and oncolytic adenovirus armed with survivin shRNA and TRAIL.     

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.     

Adenoviral gene delivery to primary human cutaneous cells and burn wounds

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.     

Tumor antigen LRRC15 impedes adenoviral infection: implications for virus-based cancer therapy

Adenoviruses for gene or oncolytic therapy are under development. Notable among these strategies is adenoviral delivery of the tumor suppressor p53. Since all therapeutics have limitations in certain settings, we have undertaken retroviral suppressor screens to identify genes conferring resistance to adenovirus-delivered p53. These studies identified the tumor antigen LRRC15, which is frequently overexpressed in multiple tumor types, as a repressor of cell death due to adenoviral p53. LRRC15, however, does not impede p53 function per se but impedes adenoviral infection. Specifically, LRRC15 causes redistribution of the coxsackievirus-adenovirus receptor away from the cell surface. This effect is manifested in less adenoviral binding to the surfaces of LRRC15-expressing cells. This discovery, therefore, not only is important for understanding adenoviral biology but also has potentially important implications for adenovirus-based anticancer therapeutics.     

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.