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Lu SC, Barry MA. Locked and loaded: engineering and arming oncolytic adenoviruses to enhance anti-tumor immune responses. Expert Opin Biol Ther 2022; 22:1359-1378. [DOI: 10.1080/14712598.2022.2139601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/04/2022]
Affiliation(s)
| | - Michael A Barry
- Division of Infectious Diseases, Department of Medicine
- Department of Immunology
- Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA
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Pfitzner S, Hofmann-Sieber H, Bosse JB, Franken LE, Grünewald K, Dobner T. Fluorescent protein tagging of adenoviral proteins pV and pIX reveals 'late virion accumulation compartment'. PLoS Pathog 2020; 16:e1008588. [PMID: 32584886 PMCID: PMC7343190 DOI: 10.1371/journal.ppat.1008588] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2020] [Revised: 07/08/2020] [Accepted: 04/30/2020] [Indexed: 12/11/2022] Open
Abstract
The human adenovirus type 5 (HAdV5) causes disease of the upper and lower respiratory tract. The early steps of HAdV5 entry up to genome replication in the host nucleus have been extensively studied. However, late stages of infection remain poorly understood. Here, we set out to elucidate the spatiotemporal orchestration of late adenovirus nuclear remodeling in living cells. We generated virus mutants expressing fluorescently tagged protein IX (pIX) and protein V (pV), a capsid and viral genome associated protein, respectively. We found that during progeny virion production both proteins localize to a membrane-less, nuclear compartment, which is highly impermeable such that in immunofluorescence microscopy antibodies can hardly penetrate it. We termed this compartment ‘late virion accumulation compartment’ (LVAC). Correlation between light- and electron microscopy revealed that the LVAC contains paracrystalline arrays of viral capsids that arrange tightly packed within a honeycomb-like organization of viral DNA. Live-cell microscopy as well as FRAP measurements showed that the LVAC is rigid and restricts diffusion of larger molecules, indicating that capsids are trapped inside. Understanding the regulation of adenovirus morphogenesis is not only of interest to cell biologists but is also key to define novel drug targets as well as to optimize adenoviruses as tools for gene therapy. While early steps of the adenovirus ‘life cycle’ are well understood, it is currently debated how, when and where capsid components associate with viral DNA. Here we used a combination of imaging methods to detail virus-induced spatiotemporal changes at late stages of infection. We found that HAdV5 induces a structured, membrane-less nuclear compartment. In this compartment capsids are closely packed within a honeycomb-like organization of replicated DNA, such that the newly formed particles appear to be trapped and show very little motility. Interestingly, we found a clear discrepancy between immunostaining and fluorescent fusion tagging, indicating a limited penetration of immunostains into this compartment. Since other pathogens induce similar compartments during replication, interpretation of immunostaining data requires careful evaluation.
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Affiliation(s)
- Søren Pfitzner
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- Center for Structural Systems Biology, Hamburg, Germany
| | - Helga Hofmann-Sieber
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
| | - Jens B. Bosse
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- Center for Structural Systems Biology, Hamburg, Germany
- RESIST Cluster of Excellence, Hannover Medical School, Germany
| | - Linda E. Franken
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- Center for Structural Systems Biology, Hamburg, Germany
| | - Kay Grünewald
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- Center for Structural Systems Biology, Hamburg, Germany
- Universität Hamburg, Institute for Biochemistry and Molecular Biology, Hamburg, Germany
| | - Thomas Dobner
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- * E-mail:
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Farrera-Sal M, Fillat C, Alemany R. Effect of Transgene Location, Transcriptional Control Elements and Transgene Features in Armed Oncolytic Adenoviruses. Cancers (Basel) 2020; 12:E1034. [PMID: 32340119 PMCID: PMC7226017 DOI: 10.3390/cancers12041034] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 04/17/2020] [Accepted: 04/21/2020] [Indexed: 12/15/2022] Open
Abstract
Clinical results with oncolytic adenoviruses (OAds) used as antitumor monotherapies show limited efficacy. To increase OAd potency, transgenes have been inserted into their genome, a strategy known as "arming OAds". Here, we review different parameters that affect the outcome of armed OAds. Recombinant adenovirus used in gene therapy and vaccination have been the basis for the design of armed OAds. Hence, early region 1 (E1) and early region 3 (E3) have been the most commonly used transgene insertion sites, along with partially or complete E3 deletions. Besides transgene location and orientation, transcriptional control elements, transgene function, either virocentric or immunocentric, and even the codons encoding it, greatly impact on transgene levels and virus fitness.
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Affiliation(s)
- Martí Farrera-Sal
- VCN Biosciences S.L., 08174 Sant Cugat, Spain
- ProCure and Oncobell Programs, Institut Català d’Oncologia/Bellbitge Biomedical Research Institute, 08908 Hospitalet de Llobregat, Spain
| | - Cristina Fillat
- August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Rare Diseases Networking Biomedical Research Center (CIBERER), University of Barcelona, 08036 Barcelona, Spain;
| | - Ramon Alemany
- ProCure and Oncobell Programs, Institut Català d’Oncologia/Bellbitge Biomedical Research Institute, 08908 Hospitalet de Llobregat, Spain
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Recombinant bovine adenovirus-3 co-expressing bovine respiratory syncytial virus glycoprotein G and truncated glycoprotein gD of bovine herpesvirus-1 induce immune responses in cotton rats. Mol Biotechnol 2015; 57:58-64. [PMID: 25173687 DOI: 10.1007/s12033-014-9801-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
Abstract
One of the impediments in the development of safe and cost effective vaccines for veterinary use has been the availability of appropriate delivery vehicle. We have chosen to develop and use bovine adenovirus (BAdV)-3 as vaccine delivery vector in cattle. Here, we describe the construction of recombinant E3 deleted BAdV-3 vectors expressing single vaccine antigen (BAV360; bovine respiratory syncytial virus G) or two vaccine antigens (BAV851; bovine herpesvirus-1gDt and bovine respiratory syncytial virus G). Recombinant proteins expressed by BAV360 or BAV851 were recognized by protein-specific monoclonal antibodies. Moreover, intranasal immunization of cotton rats with BAV360 (expressing a single vaccine antigen) or BAV851 (expressing two vaccine antigens) induced strong antigen-specific immune responses. These results suggest that single replication-competent BAdV-3 expressing vaccine antigens of two economically important respiratory pathogens of calves has potential to act as a feasible approach in the development of economically effective veterinary vaccines for cattle.
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Bauzon M, Hermiston T. Armed therapeutic viruses - a disruptive therapy on the horizon of cancer immunotherapy. Front Immunol 2014; 5:74. [PMID: 24605114 PMCID: PMC3932422 DOI: 10.3389/fimmu.2014.00074] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2013] [Accepted: 02/11/2014] [Indexed: 12/17/2022] Open
Abstract
For the past 150 years cancer immunotherapy has been largely a theoretical hope that recently has begun to show potential as a highly impactful treatment for various cancers. In particular, the identification and targeting of immune checkpoints have given rise to exciting data suggesting that this strategy has the potential to activate sustained antitumor immunity. It is likely that this approach, like other anti-cancer strategies before it, will benefit from co-administration with an additional therapeutic and that it is this combination therapy that may generate the greatest clinical outcome for the patient. In this regard, oncolytic viruses are a therapeutic moiety that is well suited to deliver and augment these immune-modulating therapies in a highly targeted and economically advantageous way over current treatment. In this review, we discuss the blockade of immune checkpoints, how oncolytic viruses complement and extend these therapies, and speculate on how this combination will uniquely impact the future of cancer immunotherapy.
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Affiliation(s)
- Maxine Bauzon
- Bayer HealthCare, US Innovation Center, Biologics Research , San Francisco, CA , USA
| | - Terry Hermiston
- Bayer HealthCare, US Innovation Center, Biologics Research , San Francisco, CA , USA
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Choi IK, Yun CO. Recent developments in oncolytic adenovirus-based immunotherapeutic agents for use against metastatic cancers. Cancer Gene Ther 2013; 20:70-6. [PMID: 23306610 DOI: 10.1038/cgt.2012.95] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
Recurrent or metastatic cancer in most cases remains an incurable disease, and thus alternative treatment strategies, such as oncolytic virotherapy, are of great interest for clinical application. Oncolytic adenoviruses (Ads) have many advantages as virotherapeutic agents and have been safely employed in the clinics. However, the efficacy of oncolytic Ads is insufficient to eradicate tumors and current clinical applications are restricted to local administration against primary tumors because of immunological obstacles and poor tumor-cell targeting. Thus, alternative viable approaches are needed to establish therapies based on oncolytic Ad that will eliminate both primary and metastatic cancers. To this end, rational design of oncolytic Ads that express immunostimulatory genes has been employed. Even when restricted to local viral delivery, these oncolytic Ad-based immunotherapeutics have been shown to exert systemic antitumor immunity and result in eradication of both primary and metastatic cancers. Moreover, oncolytic Ad-based immunotherapeutics in combination with either dendritic cell-based vaccine or radiotherapy further strengthen the systemic tumor-specific immunity, resulting in complete suppression of both local and distant tumor metastatic growth. This review will focus on the most recent updates in strategies to develop potent oncolytic Ad-based immunotherapeutics for use in cancer gene therapy.
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Affiliation(s)
- I-K Choi
- Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Korea
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7
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Oncolytic viruses: the power of directed evolution. Adv Virol 2011; 2012:586389. [PMID: 22312363 PMCID: PMC3265225 DOI: 10.1155/2012/586389] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2011] [Accepted: 05/26/2011] [Indexed: 12/17/2022] Open
Abstract
Attempts at developing oncolytic viruses have been primarily based on rational design. However, this approach has been met with limited success. An alternative approach employs directed evolution as a means of producing highly selective and potent anticancer viruses. In this method, diverse viruses are grown under conditions that maximize diversity and then passaged under conditions meant to mimic those encountered in the human cancer microenvironment. Viruses which evolve to thrive under this selective pressure are isolated and tested to identify those with increased potency (i.e., ability to replicate and spread) and/or an increased therapeutic window (i.e., differentiated replication and spread on tumor versus normal cells), both of which have potential value but the latter of which defines an oncolytic virus. Using ColoAd1, an oncolytic virus derived by this approach as a prototype, we highlight the benefits of directed evolution, discuss methods to “arm” these novel viruses, and introduce techniques for their genetic modulation and control.
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He Q, Liu Y, Zou Q, Guan YS. Transarterial injection of H101 in combination with chemoembolization overcomes recurrent hepatocellular carcinoma. World J Gastroenterol 2011; 17:2353-5. [PMID: 21633603 PMCID: PMC3098405 DOI: 10.3748/wjg.v17.i18.2353] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2010] [Revised: 02/15/2011] [Accepted: 02/22/2011] [Indexed: 02/06/2023] Open
Abstract
Transcatheter arterial chemoembolization (TACE) has become the standard treatment modality for unresectable hepatocellular carcinoma (HCC). Nonetheless, the clinical outcomes in patients with unresectable HCC are often unsatisfactory, especially in those with recurrent HCC. H101, an E1B gene deleted adenovirus, is known to have a significant antitumor activity. In addition, local injection of H101 can enhance the effect of antitumor therapies (chemotherapy and radiotherapy). Transarterial H101 gene injection in combination with TACE may help to control refractory and recurrent HCC. In this study, we report a 55-year-old patient with recurrent HCC which was treated with transarterial injection of H101 in combination with TACE, leading to a good clinical prognosis of the patient.
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Ying B, Toth K, Spencer JF, Meyer J, Tollefson AE, Patra D, Dhar D, Shashkova EV, Kuppuswamy M, Doronin K, Thomas MA, Zumstein LA, Wold WSM, Lichtenstein DL. INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies. Cancer Gene Ther 2009; 16:625-37. [PMID: 19197322 DOI: 10.1038/cgt.2009.6] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.
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Affiliation(s)
- B Ying
- VirRx Inc., St Louis, MO 63108, USA
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Abstract
Conditionally replicating adenoviruses (CRAds) have many advantages as agents for cancer virotherapy and have been safely used in human clinical trials. However, replicating adenoviruses have been limited in their ability to eliminate tumors by oncolysis. Thus, the efficacy of these agents must be improved. To this end, CRAds have been engineered to express therapeutic transgenes that exert antitumor effects independent of direct viral oncolysis. These transgenes can be expressed under native gene control elements, in which case placement within the genome determines the expression profile, or they can be controlled by exogenous promoters. The therapeutic transgenes used to arm replicating adenoviruses can be broadly classified into three groups. There are those that mediate killing of the infected cell, those that modulate the tumor microenvironment and those with immunomodulatory functions. Overall, the studies to date in animal models have shown that arming a CRAd with a rationally chosen therapeutic transgene can improve its antitumor efficacy over that of an unarmed CRAd. However, a number of obstacles must be overcome before the full potential of armed CRAds can be realized in the human clinical context. Hence, strategies are being developed to permit intravenous delivery to disseminated cancer cells, overcome the immune response and enable in vivo monitoring of the biodistribution and activity of armed CRAds.
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Affiliation(s)
- J J Cody
- Division of Human Gene Therapy, Department of Medicine, Gene Therapy Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA
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He LF, Gu JF, Tang WH, Fan JK, Wei N, Zou WG, Zhang YH, Zhao LL, Liu XY. Significant antitumor activity of oncolytic adenovirus expressing human interferon-beta for hepatocellular carcinoma. J Gene Med 2009; 10:983-92. [PMID: 18618506 DOI: 10.1002/jgm.1231] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Human interferon-beta (IFN-beta) has been widely used in gene therapy for its antitumor activity but its therapeutic effect is limited. The conditionally replicative adenovirus ONYX-015 (a E1B-55-kDa-deleted adenovirus) targets well to tumor cells, but is not potent enough to cause significant tumor regression. To solve these problems, a tumor-selective replicating adenovirus expressing IFN-beta was constructed in this study. METHODS The oncolytic adenoviruses were generated by homologous recombination in packaging cells. The expression of the IFN-beta protein was detected by enzyme-linked immunosorbent assay (ELISA). The antitumor efficacy of ZD55-IFN-beta was evaluated in cell lines and human hepatocellular carcinoma xenografts in nude mice. RESULTS ZD55-IFN-beta can express much more IFN-beta than Ad-IFN-beta because of the replication of the ZD55 vector. Our data showed that ZD55-IFN-beta could exert a strong cytopathic effect on tumor cells (about 100-fold higher than Ad-IFN-beta or ONYX-015). Moreover, no obvious cytotoxic or apoptotic effects were detected in normal cells infected with ZD55-IFN-beta. CONCLUSIONS The antitumor efficacy of IFN-beta could be significantly improved due to the increased gene expression level from the tumor-selective replicating vector. The oncolytic adenovirus expressing IFN-beta may provide a novel approach for cancer gene therapy.
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Affiliation(s)
- Ling Feng He
- Laboratory of Cancer Therapy, Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA targeting Ki-67 encoding mRNA. Cancer Gene Ther 2008; 16:20-32. [PMID: 18690204 DOI: 10.1038/cgt.2008.61] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
RNA interference (RNAi) has been proved to be a powerful tool for gene knockdown purpose and holds great promise for the treatment of cancer. Our previous study demonstrated that the reduction of Ki-67 expression by means of chemically synthesized siRNAs and shRNAs expressed from plasmid resulted in proliferation inhibition in human renal carcinoma cells. In this study, we constructed a novel oncolytic adenovirus-based shRNA expression system, ZD55-Ki67, and explored ZD55-Ki67-mediated RNAi for Ki-67 gene silencing. Our results showed that ZD55-Ki67 could induce silencing of the Ki-67 gene effectively, allow for efficient tumor-specific viral replication and induce the apoptosis of tumor cells effectively in vitro and in nude mice. We conclude that combining shRNA gene therapy and oncolytic virotherapy can enhance antitumor efficacy as a result of synergism between CRAd oncolysis and shRNA antitumor responses.
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Luo J, Xia Q, Zhang R, Lv C, Zhang W, Wang Y, Cui Q, Liu L, Cai R, Qian C. Treatment of Cancer with a Novel Dual-Targeted Conditionally Replicative Adenovirus Armed with mda-7/IL-24 Gene. Clin Cancer Res 2008; 14:2450-7. [DOI: 10.1158/1078-0432.ccr-07-4596] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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An empty E1, E3, E4 adenovirus vector protects photoreceptors from light-induced degeneration. J Ocul Biol Dis Infor 2008; 1:30-6. [PMID: 20072633 PMCID: PMC2802415 DOI: 10.1007/s12177-008-9004-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2008] [Accepted: 03/12/2008] [Indexed: 11/30/2022] Open
Abstract
We have previously identified a neuroprotective effect associated with empty (E1−, E3−, E4−) adenovirus vector delivery in a model of light-induced, photoreceptor cell death. In this study, we further characterize this protective effect in light-injured retina and investigate its molecular basis. Dark-adapted BALB/c mice, aged 6–8 weeks, were exposed to standardized, intense fluorescent light for 96 or 144 h. Prior to dark adaptation, all mice received intravitreous injection of 1 × 109 particles of an empty (E1−, E3−, E4−) adenovirus vector in one eye and vehicle in the other. Following light challenge of 96 or 144 h, histopathological analysis and quantitative photoreceptor cell counts were conducted. Semiquantitative assessment of messenger ribonucleic acid (mRNA) for the apoptosis related genes: p50, p65, IkBa, caspase-1, caspase-3, Bad, c-Jun, Bax, Bak, Bcl-2, c-Fos, and p53 using quantitative reverse transcriptase polymerase chain reaction was performed on eyes following 12 h of light exposure. Following 96 h of light exposure, the photoreceptor cell density for E1−, E3−, E4− adenovirus vector and vehicle-injected eyes were 87.5 ± 9.5 and 79.3 ± 10.1, respectively, (p = 0.79). After 144 h of light exposure, the photoreceptor cell density was preserved in vector-injected eyes as compared to vehicle treated eyes, 68.9 ± 10.0 and 49.2 ± 4.6, respectively (p = 0.016). Relative mRNA levels of c-Fos and c-Jun at 12-h light exposure after injection differed significantly between vector- and vehicle-injected eyes (p = 0.036, 0.016, respectively). The expression of the other apoptosis-related genes evaluated was not significantly affected. This study investigates the molecular basis of photoreceptor neuroprotective pathway induction associated with E1−, E3−, E4− adenovirus vectors. The results indicate that empty adenovirus vectors protect photoreceptors from light-induced degeneration by the modulation of apoptotic pathways. Gene expression changes suggest that the suppression of c-Fos and c-Jun upregulation contributes significantly to the neuroprotective effect. Understanding the molecular basis of the neuroprotective pathway induction in photoreceptors is critical to the development of novel therapies for retinal degenerations.
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Vogels R, Zuijdgeest D, van Meerendonk M, Companjen A, Gillissen G, Sijtsma J, Melis I, Holterman L, Radosevic K, Goudsmit J, Havenga MJE. High-level expression from two independent expression cassettes in replication-incompetent adenovirus type 35 vector. J Gen Virol 2007; 88:2915-2924. [PMID: 17947512 DOI: 10.1099/vir.0.83119-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Replication-incompetent adenovirus type 35 (rAd35) represents a potent vaccine carrier that elicits strong, antigen-specific T- and B-cell responses in diverse preclinical models. Moreover, Ad35 is rare in human populations, resulting in the absence of neutralizing antibodies against this carrier, in contrast to the commonly used rAd5. Therefore, rAd35 is being investigated as a vaccine carrier for a number of diseases for which an effective vaccine is needed, including malaria, AIDS and tuberculosis. However, it can be perceived that effective immunization will require insertion of multiple antigens into adenoviral vectors. We therefore wanted to create rAd35 vectors carrying double expression cassettes, to expand within one vector the number of insertion sites for foreign DNA encoding antigenic proteins. We show that it is possible to generate rAd35 vectors carrying two cytomegalovirus promoter-driven expression cassettes, provided that the polyadenylation signals in each expression cassette are not identical. We demonstrate excellent rAd35 vector stability and show that expression of a transgene is not influenced by the presence of a second expression cassette. Moreover, by using two model vaccine antigens, i.e. the human immunodeficiency virus-derived Env-gp120 protein and the Plasmodium falciparum-derived circumsporozoite protein, we demonstrate that potent T- and B-cell responses are induced to both antigens expressed from a single vector. Such rAd35 vectors thus expand the utility of rAd35 vaccine carriers for the development of vaccines against, for example, malaria, AIDS and tuberculosis.
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Affiliation(s)
- Ronald Vogels
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | - David Zuijdgeest
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | | | - Arjen Companjen
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | - Gert Gillissen
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | - Jeroen Sijtsma
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | - Irene Melis
- Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
| | | | | | - Jaap Goudsmit
- Center of Poverty-Related Communicable Diseases, Academic Medical Center, Amsterdam, The Netherlands.,Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
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Robinson M, Ge Y, Ko D, Yendluri S, Laflamme G, Hawkins L, Jooss K. Comparison of the E3 and L3 regions for arming oncolytic adenoviruses to achieve a high level of tumor-specific transgene expression. Cancer Gene Ther 2007; 15:9-17. [PMID: 17853920 DOI: 10.1038/sj.cgt.7701093] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Arming oncolytic adenoviral vectors with anticancer transgenes that can be expressed in a tumor-selective manner may enable the engineering of vectors with increased potency, while retaining their safety profile. Armed oncolytic adenoviral vectors were constructed in which transgene expression has been linked via modified splice acceptor sequences that did not necessitate the deletion of any part of the adenoviral genome. Several oncolytic adenoviral vectors were compared in which the transgene was inserted in place of either the E3 or the L3 region. While all vectors had similar viral growth and cytotoxicity characteristics, the highest level of transgene expression was observed from a vector in which the transgene had been inserted downstream of the L3 23K protease gene, the Ad-23K-GM vector. Notably, no transgene expression occurred with this vector in the absence of DNA replication either in vitro or in vivo. In contrast, viruses in which the transgene was inserted into E3 locations exhibited a low level of transgene expression even in the absence of DNA replication. In summary, by utilizing the L3 region for arming oncolytic viruses, higher levels of tumor-specific transgene expression can be obtained without the need to delete any parts of the viral genome.
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Affiliation(s)
- M Robinson
- Cell Genesys Inc., South San Francisco, California, CA 94080, USA.
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Kretschmer PJ, Jin F, Chartier C, Hermiston TW. Development of a transposon-based approach for identifying novel transgene insertion sites within the replicating adenovirus. Mol Ther 2006; 12:118-27. [PMID: 15963927 DOI: 10.1016/j.ymthe.2005.03.019] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2005] [Revised: 03/21/2005] [Accepted: 03/21/2005] [Indexed: 10/25/2022] Open
Abstract
Therapeutic gene delivery from an oncolytic adenovirus (Ad) is one approach to enhancing the potency of Ad-based virotherapies for cancer. To identify therapeutic transgene insertion sites compatible with the replicating virus, a methodology that broadly scans the viral genome is needed. To address this we modified a transposon (Tn7)-based in vitro transposition system to take advantage of its nonprejudiced scanning ability to identify insertion sites compatible with viral replication. Using this system with a plasmid containing an E3-deleted Ad5, we identified several unique sites for promoter-based expression cassette insertions within the Ad genome. The transposon-based expression cassette is bounded by PmeI restriction endonuclease sites unique to the transposon, making expression cassette substitutions easy to perform. Additional expression cassettes containing different promoters and reporter genes were substituted into two of the newly identified transgene insertion sites. The results suggest that the ease and orientation of expression cassette substitution depend on both the insertion site location and the promoter and gene of the replacement expression cassette. These studies establish the transposon-based system as an efficient approach to scanning the Ad genome and identifying insertion sites compatible with viral replication and represents a powerful tool for the development of armed therapeutic viruses for cancer.
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Affiliation(s)
- Peter J Kretschmer
- Gene Therapy Research Department, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
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Gao Q, Zhou J, Huang X, Chen G, Ye F, Lu Y, Li K, Zhuang L, Huang M, Xu G, Wang S, Ma D. RETRACTED: Selective targeting of checkpoint kinase 1 in tumor cells with a novel potent oncolytic adenovirus. Mol Ther 2006; 13:928-937. [PMID: 16459149 DOI: 10.1016/j.ymthe.2005.12.009] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2005] [Revised: 12/15/2005] [Accepted: 12/27/2005] [Indexed: 02/04/2023] Open
Abstract
This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the editor-in-chief. Similarities were reported between images in this article and an article in Clinical Cancer Research (Zhou et al., 2005, Clin. Cancer Res. 11, 8431-8440, https://10.1158/1078-0432.CCR-05-1085). Q.J., J.Z., X.H., G.C., Y.L., K.L., L.Z., and D.M. were all authors of the Clinical Cancer Research paper as well. These concerns were initially reported in a Pubpeer thread (https://pubpeer.com/publications/FF881782FF5AFD316D42E0C0F00766). Image analysis performed by the editorial office confirmed findings of image recycling in Figures 2A and 3B of the Molecular Therapy article. This reuse (and in part misrepresentation) of data without appropriate attribution represents a severe abuse of the scientific publishing system.
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MESH Headings
- Adenoviridae/genetics
- Adenoviridae/physiology
- Adenovirus E1A Proteins/deficiency
- Adenovirus E1A Proteins/genetics
- Animals
- Antineoplastic Agents/therapeutic use
- Carcinoma, Hepatocellular/enzymology
- Carcinoma, Hepatocellular/therapy
- Carcinoma, Hepatocellular/virology
- Cell Line
- Cell Line, Tumor
- Cell Survival
- Checkpoint Kinase 1
- Cisplatin/therapeutic use
- Cytopathogenic Effect, Viral/physiology
- DNA, Antisense/genetics
- Female
- Gene Targeting
- Genetic Vectors/administration & dosage
- Genetic Vectors/therapeutic use
- HeLa Cells
- Humans
- Liver Neoplasms/enzymology
- Liver Neoplasms/therapy
- Liver Neoplasms/virology
- Mice
- Mice, Inbred BALB C
- Mice, Nude
- Models, Genetic
- Promoter Regions, Genetic
- Protein Kinases/genetics
- Protein Kinases/metabolism
- Virus Replication
- Xenograft Model Antitumor Assays
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Affiliation(s)
- Qinglei Gao
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Jianfeng Zhou
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Xiaoyuan Huang
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Gang Chen
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Fei Ye
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Yunping Lu
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Kanyan Li
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Liang Zhuang
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Mei Huang
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Gang Xu
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Shxuan Wang
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China
| | - Ding Ma
- Cancer Biology Research Center, TongJi Hospital, TongJi Medical School, Huazhong University of Science & Technology, WuHan, Hubei 430030, People's Republic of China.
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Abstract
Advances in gene modification and viral therapy have led to the development of a variety of vectors in several viral families that are capable of replication specifically in tumor cells. Because of the nature of viral delivery, infection, and replication, this technology, oncolytic virotherapy, may prove valuable for treating cancer patients, especially those with inoperable tumors. Current limitations exist, however, for oncolytic virotherapy. They include the body's B and T cell responses, innate inflammatory reactions, host range, safety risks involved in using modified viruses as treatments, and the requirement that most currently available oncolytic viruses require local administration. Another important constraint is that genetically enhanced vectors may or may not adhere to their replication restrictions in long-term applications. Several solutions and strategies already exist, however, to minimize or circumvent many of these limitations, supporting viral oncolytic therapy as a viable option and powerful tool in the fight against cancer.
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Affiliation(s)
- J J Davis
- Department of Thoracic and Cardiovascular Surgery, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
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20
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21
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Shen Y, Senzer N, Nemunaitis J. Individualised cancer therapeutics: dream or reality? Therapeutics construction. Expert Opin Biol Ther 2005; 5:1427-41. [PMID: 16255647 DOI: 10.1517/14712598.5.11.1427] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The analysis of DNA microarray and proteomic data, and the subsequent integration into functional expression sets, provides a circuit map of the hierarchical cellular networks responsible for sustaining the viability and environmental competitiveness of cancer cells, that is, their robust systematics. These technologies can be used to 'snapshot' the unique patterns of molecular derangements and modified interactions in cancer, and allow for strategic selection of therapeutics that best match the individual profile of the tumour. This review highlights technology that can be used to selectively disrupt critical molecular targets and describes possible vehicles to deliver the synthesised molecular therapeutics to the relevant cellular compartments of the malignant cells. RNA interference (RNAi) involves a group of evolutionarily conserved gene silencing mechanisms in which small sequences of double-stranded RNA or intrinsic antisense RNA trigger mRNA cleavage or translational repression, respectively. Although RNAi molecules can be synthesised to 'silence' virtually any gene, even if upregulated, a mechanism for selective delivery of RNAi effectors to sites of malignant disease remains challenging. The authors will discuss gene-modified conditionally replicating viruses as candidate vehicles for the delivery of RNAi.
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Affiliation(s)
- Yuqiao Shen
- Mary Crowley Medical Research Center, Dallas, TX 75201, USA
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22
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Zhang Z, Zou W, Wang J, Gu J, Dang Y, Li B, Zhao L, Qian C, Qian Q, Liu X. Suppression of tumor growth by oncolytic adenovirus-mediated delivery of an antiangiogenic gene, soluble Flt-1. Mol Ther 2005; 11:553-62. [PMID: 15771958 DOI: 10.1016/j.ymthe.2004.12.015] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2004] [Accepted: 12/24/2004] [Indexed: 12/14/2022] Open
Abstract
Armed oncolytic adenoviruses represent an appealing tumor treatment approach, as they can attack tumors at multiple levels. In this study, considering that angiogenesis plays a central role in tumor growth, we inserted an antiangiogenic gene, sflt-1(1-3) (the first three extracellular domains of FLT1, the hVEGF receptor-1), into an E1B-55-kDa-deleted oncolytic adenovirus (ZD55) to construct ZD55-sflt-1. Although soluble (s) Flt-1 did not affect tumor cell growth, ZD55-sflt-1 could specifically induce a cytopathic effect in tumor cells, like ONYX-015. The secretion of sFlt-1 from ZD55-sflt-1 was much higher than that from replication-deficient Ad-sflt-1 upon infection of SW620 human colon tumor cells, leading to a stronger inhibitory effect on VEGF-induced proliferation and tube formation ability of HUVECs. Moreover, marked reduction of tumor growth and long-term survival rates were observed in ZD55-sflt-1-treated nude mice with subcutaneous SW620 tumor. Its efficacy correlated with a decrease in microvessel density and an increase in apoptotic tumor cells. In addition, ZD55-sflt-1 showed a synergic effect with the chemotherapeutic agent 5-FU. These results indicate that ZD55-sflt-1, combining the advantages of oncolytic adenovirus and antiangiogenic gene therapy, is a powerful agent for human tumor treatment.
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Affiliation(s)
- Zilai Zhang
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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23
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Jin F, Kretschmer PJ, Hermiston TW. Identification of novel insertion sites in the Ad5 genome that utilize the Ad splicing machinery for therapeutic gene expression. Mol Ther 2005; 12:1052-63. [PMID: 16165398 DOI: 10.1016/j.ymthe.2005.07.696] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2005] [Revised: 07/28/2005] [Accepted: 07/28/2005] [Indexed: 11/18/2022] Open
Abstract
Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics. In the case of the oncolytic adenovirus (Ad), however, the genomic packaging capacity is constrained. To address this, we explored whether a transposon-based system could identify sites in the viral genome where endogenous Ad promoters could drive transgene expression via splicing and still maintain the replication capacity of the virus. Using GFP as a reporter gene and an E3-deleted Ad genome as a target, we tested three splicing signals. RACE analysis confirmed that gene expression from the GFP-expressing Ads occurs via splicing and traced expression to the Ad major late promoter (MLP). Replacement of the GFP transposon by an equivalent splice acceptor-luciferase expression cassette in the same orientation confirmed that substitute transgenes are also expressed via splicing from the MLP. Interestingly, insertion of the substitute transgene in the opposite orientation also resulted in expression that, in some cases, originated from within the ITR region of the viral genome. In summary, splice acceptor sequences can be used to control transgene expression from endogenous Ad promoters and this represents a genomically economical approach to arming oncolytic Ads.
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Affiliation(s)
- Fang Jin
- Gene Therapy Research Department, Berlex Biosciences, 2600 Hilltop Drive, Richmond, CA 94804, USA
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24
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Zhu M, Bristol JA, Xie Y, Mina M, Ji H, Forry-Schaudies S, Ennist DL. Linked tumor-selective virus replication and transgene expression from E3-containing oncolytic adenoviruses. J Virol 2005; 79:5455-65. [PMID: 15827160 PMCID: PMC1082742 DOI: 10.1128/jvi.79.9.5455-5465.2005] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
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.
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Affiliation(s)
- Mingzhu Zhu
- Immunology Program, Oncology Unit, Genetic Therapy, Inc., Bethesda, MD 20817, USA
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25
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Carette JE, Graat HCA, Schagen FHE, Abou El Hassan MAI, Gerritsen WR, van Beusechem VW. Replication-dependent transgene expression from a conditionally replicating adenovirus via alternative splicing to a heterologous splice-acceptor site. J Gene Med 2005; 7:1053-62. [PMID: 15756711 DOI: 10.1002/jgm.754] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
BACKGROUND Oncolytic viruses are promising anticancer agents because they selectively kill cancer cells and multiply within a tumor. Their oncolytic potency might be improved by expressing a therapeutic gene from the virus genome. In this regard, proper kinetics and level of transgene expression are important. In addition, expression of cytotoxic transgene products should be confined to cancer cells. Here, we developed oncolytic adenoviruses that provide transgene expression dependent on viral replication. METHODS We constructed an oncolytic adenovirus that expresses luciferase under regulation of the endogenous major late promoter (MLP) via alternative splicing to an inserted splice-acceptor site analogous to that of the adenovirus serotype 40 long fiber gene. Splicing of the luciferase transcript was studied by RT-PCR analysis. Expression was measured in the presence and absence of the flavonoid apigenin, an inhibitor of viral replication. RESULTS The inserted splice-acceptor site was properly recognized by the adenoviral splicing machinery. Luciferase expression levels were markedly higher than levels obtained with the cytomegalovirus (CMV) promoter, especially at late stages of infection. Inhibiting adenovirus replication reduced luciferase expression levels dramatically by 4 to 5 logs, whereas expression levels with the CMV-luciferase adenovirus were only moderately affected (2 logs). CONCLUSIONS Transgene delivery using the endogenous late gene expression machinery resulted in an expression pattern distinct from expression driven by the conventional CMV promoter. The high expression levels and strict coupling of expression to viral replication should be useful for adequate monitoring of replication and might provide a platform for the design of armed conditionally replicating adenoviruses (CRAds) with enhanced oncolytic potency.
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Affiliation(s)
- Jan E Carette
- Division of Gene Therapy, Department of Medical Oncology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands.
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26
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Lu W, Zheng S, Li XF, Huang JJ, Zheng X, Li Z. Intra-tumor injection of H101, a recombinant adenovirus, in combination with chemotherapy in patients with advanced cancers: A pilot phase II clinical trial. World J Gastroenterol 2004; 10:3634-8. [PMID: 15534920 PMCID: PMC4612006 DOI: 10.3748/wjg.v10.i24.3634] [Citation(s) in RCA: 90] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: H101, an E1B 55 kD gene deleted adenovirus, has been shown to possess oncolysis activity experimentally and proved to be safe in preliminary phase I study. The current study was designed to evaluate its anti-tumor activity and toxicity in combination with chemotherapy in patients with late stage cancers.
METHODS: H101 5.0 × 1011 virus particles were given by intra-tumor injection daily for five consecutive days at every three-week cycle, combined with routine chemotherapy, to one of the tumor lesions of 50 patients with different malignant tumors. Tumor lesions without H101 injection in the same individuals were used as controls. The efficacy and toxicity were recorded.
RESULTS: Forty-six patients were evaluable with a 30.4% response rate. H101 injection in combination with chemotherapy induced three complete response (CR) and 11 partial response (PR), giving an overall response rate of 28.0% (14/50) among intention-to-treat patients. The response rate for the control lesions was 13.0%, including one case with CR and five cases with PR, which was significantly lower than that for the injected lesions (P < 0.05). Main side effects were fever (30.2%) and pain at the injected sites (26.9%). Grade 1 hepatic dysfunction was found in four patients, grade 2 in one patient, and grade 4 in one patient. Hematological toxicity (grade 4) was found in four patients.
CONCLUSION: Intra-tumor injection of the genetically engineered adenovirus H101 exhibits potential anti-tumor activity to refractory malignant tumors in combination with chemotherapy. Low toxicity and good tolerance of patients to H101were observed.
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Affiliation(s)
- Wei Lu
- Cancer Center, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jiefang Road, Hangzhou 310009, Zhejiang Province, China
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27
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Zhan J, Gao Y, Wang W, Shen A, Aspelund A, Young M, Laquerre S, Post L, Shen Y. Tumor-specific intravenous gene delivery using oncolytic adenoviruses. Cancer Gene Ther 2004; 12:19-25. [PMID: 15514685 DOI: 10.1038/sj.cgt.7700730] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
In this report, we describe a vector system that specifically delivers transgene products to tumors following intravenous (i.v.) administration. The Escherichia coli cytosine deaminase (CD) gene was placed in the E3B region of the tumor-selective, replication-competent adenovirus ONYX-411, under the control of endogenous viral late gene regulatory elements. Thus, CD expression was directly coupled to the tumor-selective replication of the viral vector. In vitro, CD was expressed efficiently in various human cancer cell lines tested but not in cultured normal human cells, including human hepatocytes. Following i.v. administration into nude mice carrying human tumor xenografts, robust CD activity was detected only in tumors but not in liver or other normal tissues. Levels of CD activity in the tumors increased progressively following i.v. virus administration, correlating closely with virus replication in vivo. Subsequent administration of 5-fluorocytosine (5-FC) demonstrated a trend to improve the antitumor efficacy of these viruses in a mouse xenograft model, presumably due to the intratumoral conversion of 5-FC to the chemotherapeutic drug 5-fluorouracil. We show that the combination of a highly selective oncolytic virus, ONYX-411, with the strategic use of the viral E3B region for transgene insertion provides a powerful platform that allows for tumor-specific, persistent and robust transgene expression after i.v. administration. This technology provides an opportunity to enhance greatly both safety and efficacy of cancer gene therapy.
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Affiliation(s)
- Jinghui Zhan
- Onyx Pharmaceuticals Inc., Richmond, California 94806, USA
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28
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Lee CT, Park KH, Yanagisawa K, Adachi Y, Ohm JE, Nadaf S, Dikov MM, Curiel DT, Carbone DP. Combination Therapy with Conditionally Replicating Adenovirus and Replication Defective Adenovirus. Cancer Res 2004; 64:6660-5. [PMID: 15374981 DOI: 10.1158/0008-5472.can-04-1200] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.
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Affiliation(s)
- Choon-Taek Lee
- Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37322-6838, USA
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29
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Fuerer C, Iggo R. 5-Fluorocytosine increases the toxicity of Wnt-targeting replicating adenoviruses that express cytosine deaminase as a late gene. Gene Ther 2004; 11:142-51. [PMID: 14712298 DOI: 10.1038/sj.gt.3302148] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Clinical studies with oncolytic adenoviruses have shown that existing viruses are safe but lack efficacy. To selectively increase the toxicity of oncolytic adenoviruses targeting colon tumours, we have inserted the yeast cytosine deaminase gene (yCD) after the fibre gene in the major late transcript. yCD was expressed using either an internal ribosome entry site (IRES) or by alternative splicing of a new exon analogous to the Ad41 long fibre exon. The IRES-CD virus gave higher yCD expression on Western blots. Both approaches result in yCD expression restricted to the period after viral DNA replication. Viral burst size was reduced by less than approximately 10-fold by 5-fluorocytosine (5-FC), showing that expression of yCD as a late gene is compatible with virus replication. Cytopathic effect assays in colon cancer cell lines showed that both yCD viruses have approximately 10-fold increased toxicity in the presence of the prodrug 5-FC, which is converted to 5-fluorouracil (5-FU) by yCD. Toxicity was higher following addition of 5-FC immediately after infection. The largest gain in toxicity was seen in HT29 colon cancer cells, which are the least permissive colon cancer cells for the parental virus, indicating that the new 5-FC/yCD viruses may have broader applications for colon cancer therapy than their predecessors.
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Affiliation(s)
- C Fuerer
- Oncogene Group, NCCR Molecular Oncology, Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland
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30
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Abstract
An international meeting was held on the development and application of replicating viruses for cancer therapy this past March in Banff, Alberta. In this review, using the presentations at this meeting as a backdrop, we discuss how recent scientific and clinical findings are reshaping the development of oncolytic virus therapeutics. Here we identify some of the obstacles that these therapeutics face and discuss evolving strategies, both preclinically and clinically, that are facilitating oncolytic virus development.
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Affiliation(s)
- John C Bell
- Ottawa Regional Cancer Centre, 503 Smyth Road, Ottawa, Ontario K1H 1C4, Canada.
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31
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Affiliation(s)
- Karoly Toth
- Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104, USA
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