The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Hirofumi Hamada - One of the best experts on this subject based on the ideXlab platform.
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cyclophosphamide enhances antitumor efficacy of oncolytic adenovirus expressing Uracil Phosphoribosyltransferase uprt in immunocompetent syrian hamsters
International Journal of Cancer, 2013Co-Authors: Naoyuki Hasegawa, Hirofumi Hamada, Masato Abei, Kazunari K Yokoyama, Kuniaki Fukuda, Rei Kawashima, Yuri Nakano, Takeshi Yamada, Koji Nakade, Yuichi ObataAbstract:Oncolytic viruses (OVs) are novel cancer therapeutics with great promise, but host antiviral immunity represents the hurdle for their efficacy. Immunosuppression by cyclophosphamide (CP) has thus been shown to enhance the oncolytic efficacy of many OVs, but its effects on OVs armed with therapeutic genes remain unknown. We have previously reported on the efficacy of AxE1CAUP, an oncolytic adenovirus (OAd) expressing Uracil Phosphoribosyltransferase (UPRT), an enzyme that markedly enhanced the toxicity of 5-fluoroUracil (5-FU), in immunodeficient, Ad-nonpermissive nude mice. Here we explored the efficacy and safety of intratumoral (i.t.) AxE1CAUP/5-FU therapy and of its combination with CP for syngenic HaP-T1 pancreatic cancers in immunocompetent, Ad-permissive Syrian hamsters. AxE1CAUP infected, replicated, expressed UPRT, and increased the sensitivity to 5-FU in HaP-T1 cells in vitro. I.t. AxE1CAUP/5-FU treatment inhibited the growth of subcutaneous HaP-T1 allografts. The combination with high-dose CP inhibited serum Ad-neutralizing antibody formation, increased intratumoral AxE1CAUP replication and UPRT expression, and resulted in further enhanced therapeutic effects with 5-FU. Neither body weight nor histology of the liver and lung changed during these treatments. A clinically-approved, intermediate-dose CP also enhanced the efficacy of i.t. AxE1CAUP/5-FU treatment in these hamsters, which was not affected by preexisting immunity to the vector. These data demonstrate the excellent antitumor efficacy and safety of an OAd armed with a suicide gene in combination with CP for treating syngenic tumors in immunocompetent, Ad-permissive animals, indicating the efficacy of CP in overcoming the hurdle of antiviral immunity for effective OV-mediated gene therapy.
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effective gene therapy of biliary tract cancers by a conditionally replicative adenovirus expressing Uracil Phosphoribosyltransferase significance of timing of 5 fluoroUracil administration
Cancer Research, 2005Co-Authors: Masato Abei, Hirofumi Hamada, Kuniaki Fukuda, Mariko Wakayama, Hideyo Ugai, Takehide Murata, Takeshi Todoroki, Yasushi Matsuzaki, Naomi Tanaka, Kazunari K YokoyamaAbstract:In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for Uracil Phosphoribosyltransferase (UPRT), which converts 5-fluoroUracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.
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chemo radio gene therapy for colorectal cancer cells using escherichia coli Uracil Phosphoribosyltransferase gene
Anticancer Research, 2003Co-Authors: Fumikazu Koyama, Hirofumi Hamada, Hisao Fujii, Tomohide Mukogawa, Masato Ueno, Hirofumi Ishikawa, Takeshi Nakao, Hiroshi Matsumoto, Hidehiko Shimatani, Taku TakeuchiAbstract:: 5-FluoroUracil (5-FU) is one of the most widely used chemotherapeutic agents, and is known to be a radiosensitizer. Previously, we reported that adenoviral transduction of the Escherichia coli (E. coli) Uracil Phosphoribosyltransferase (UPRT) gene induced marked sensitivity in human colon cancer cells to 5-FU. The aim of the current study was to investigate the efficacy of virally-directed UPRT and 5-FU to enhance the radiosensitivity of HT29 human colon cancer cells. Cytotoxicity as a result of radiation treatment following AdCA-UPRT infection and 5-FU exposure was confirmed by radiation dose-response analysis with colony formation assay. In vivo chemoradio-gene therapy using the UPRT/5-FU/radiation system showed tumor regressive effects even against large HT29-established subcutaneous tumors in nude mice. Our results suggested that adenovirus-mediated UPRT gene transduction combined with 5-FU administration and radiation may be an effective new chemo-radio-gene therapy for colorectal cancer.
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combined radiation and gene therapy for brain tumors with adenovirus mediated transfer of cytosine deaminase and Uracil Phosphoribosyltransferase genes
Cancer Gene Therapy, 2002Co-Authors: Hirokazu Kambara, Hirofumi Hamada, Takashi Tamiya, Shinji Ohtsuka, Kinya Terada, Yoshiaki Adachi, Tomotsugu Ichikawa, Takashi OhmotoAbstract:Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)/cytosine deaminase (CD) plus Uracil Phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic interaction between radiation therapy and 5-FC/CD+UPRT gene therapy for glioma cells. In vivo experiments demonstrated that the combination of gene therapy and radiation possessed superior antitumor effect in comparison to single therapy. However, the adverse effects of radiation therapy in combination with the gene therapy were observed with respect to normal brain. This combination therapy may be feasible for the treatment of gliomas, although the radiation dose and area should be reduced in order to prevent side effects.
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adenovirus mediated transduction of escherichia coli Uracil Phosphoribosyltransferase gene increases the sensitivity of esophageal cancer cells to 5 fluoroUracil
Surgery Today, 2001Co-Authors: Hajime Nakamura, Hirofumi Hamada, Hiroyuki Sekiguchi, Seiji Akiyama, Michitaka Fujiwara, Yasushi Kasai, Akimasa NakaoAbstract:Esophageal cancer is associated with the poorest prognosis among the digestive tract cancers, and chemotherapy is the treatment of choice for many patients. In this study, we experimentally introduced an Escherichia coli-derived Uracil Phosphoribosyltransferase (UPRT) gene to cultured esophageal cancer cell lines to potentiate the antitumor effects of a representative anticancer drug, 5-fluorouacil (5-FU). UPRT is a pyrimidine salvage enzyme that catalyzes the synthesis of uridine monophosphate from Uracil and PRPP. The UPRT gene was transduced into five cultured esophageal cancer cell lines, TE1, TE2, TE3, NUEC1, and T.T, using an adenovirus vector. It was confirmed that the sensitivities of all cultured cell lines to 5-FU were increased in vitro. Subsequently, the T.T line was subcutaneously inoculated into nude mice to induce tumors, after which 5-FU was administered intraperitoneally. When a UPRT gene-recombinant adenovirus vector was directly injected into the tumors, tumor proliferation was markedly inhibited compared with that in the group treated with 5-FU alone, suggesting potentiation of 5-FU sensitivity by UPRT gene transduction in vivo. Therefore, we potentiated the effects of commercially available anticancer drugs by gene transduction. Our method may prove useful as a new form of cancer gene therapy in the future.
Michael D Cleary - One of the best experts on this subject based on the ideXlab platform.
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TU-tagging: cell type specific RNA isolation from intact complex tissues
Nature Methods, 2009Co-Authors: Michael R. Miller, Kristin J. Robinson, Michael D ClearyAbstract:Expressing Uracil Phosphoribosyltransferase in specific tissues in the fly allows the incorporation of 4-thioUracil into newly synthesized RNA in vivo. The thio-labeled RNA can then be isolated and analyzed by routine procedures allowing the cell type–specific measure of RNA synthesis and decay rates.
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cell type specific analysis of mrna synthesis and decay in vivo with Uracil Phosphoribosyltransferase and 4 thioUracil
Methods in Enzymology, 2008Co-Authors: Michael D ClearyAbstract:Abstract Microarray‐based analysis of mRNA expression has provided a genome‐wide understanding of the genes and pathways involved in many biological processes. However, two limitations are often associated with traditional microarray experiments. First, standard methods of microarray analysis measure mRNA abundance, not mRNA synthesis or mRNA decay, and, therefore, do not provide any information regarding the mechanisms regulating transcript levels. Second, microarrays are often performed with mRNA from a mixed population of cells, and data for a specific cell‐type of interest can be difficult to obtain. This chapter describes a method, referred to here as “4TU‐tagging,” which can be used to overcome these limitations. 4TU‐Tagging uses cell type–specific expression of the Uracil Phosphoribosyltransferase gene of Toxoplasma gondii and the Uracil analog 4‐thioUracil (4TU) to selectively tag and purify RNA. Pulse‐labeling of newly synthesized RNA with 4TU followed by a “chase” with unmodified Uracil allows in vivo measurements of mRNA synthesis and decay in specific cells. Experimental design considerations for applying 4TU‐tagging to different systems and protocols for cell type–specific RNA tagging, purification, and microarray analysis are covered in this chapter.
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rna analysis by biosynthetic tagging using 4 thioUracil and Uracil Phosphoribosyltransferase
Methods of Molecular Biology, 2008Co-Authors: Gusti M Zeiner, Michael D Cleary, Ashley E Fouts, Christopher D Meiring, Edward S Mocarski, John C BoothroydAbstract:: RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii Uracil Phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of Uracil to yield uridine monophosphate (UMP). When 4-thioUracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.
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biosynthetic labeling of rna with Uracil Phosphoribosyltransferase allows cell specific microarray analysis of mrna synthesis and decay
Nature Biotechnology, 2005Co-Authors: Michael D Cleary, Christopher D Meiering, Rebecca Guymon, John C BoothroydAbstract:Biosynthetic labeling of RNA with Uracil Phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay
Bente Mygind - One of the best experts on this subject based on the ideXlab platform.
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different oligomeric states are involved in the allosteric behavior of Uracil Phosphoribosyltransferase from escherichia coli
FEBS Journal, 1996Co-Authors: Kaj Frank Jensen, Bente MygindAbstract:Uracil Phosphoribosyltransferase, catalyzing the formation of UMP and pyrophosphate from Uracil and 5-phosphoribosyl-α-1-diphosphate (PP RibP), was purified from an overproducing strain of Escherichia coli. GTP was shown to activate the enzyme by reducing Km for PP RibP by about fivefold without affecting Vmax. When started by addition of enzyme, the reactions accelerated over an extended period of time, while enzyme solutions incubated first with GTP and PP RibP displayed constant velocities. This indicated that PP RibP and GTP influenced the structure of the enzyme. Gel-filtration and sedimentation analyses showed that the apparent oligomeric state of Uracil Phosphoribosyltransferase is defined by a dynamic equilibrium between a slowly sedimenting form (dimeric or trimeric) that has only a little activity, and a more highly aggregated form (pentameric or hexameric), which is more active. It appears that the smaller form predominates in the absence of substrates, while the larger form predominates in the presence of GTP and PP RibP. Guanosine-3′,5′-bis(diphosphate) was found to activate the enzyme much like GTP.
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Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene.
Journal of Bacteriology, 1995Co-Authors: Paal Skytt Andersen, Dorte Frees, Robert Fast, Bente MygindAbstract:Mutants defective in utilization of Uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding Uracil Phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids.
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characterization of the upp gene encoding Uracil Phosphoribosyltransferase of escherichia coli k12
FEBS Journal, 1992Co-Authors: Paal Skytt Andersen, John M Smith, Bente MygindAbstract:The upp gene coding for Uracil Phosphoribosyltransferase was subcloned on a 5-kb EcoRI restriction fragment along with the purMN operon. By a combination of complementation, deletion and minicell analyses, the upp gene was located adjacent to and divergently transcribed from the purMN operon. All three gene products could be identified in minicell extracts. The cloned upp gene shows an elevated expression upon Uracil starvation. The nucleotide sequence and transcription start of the gene were determined. The sequence yields an open reading frame of 624 nucleotides encoding a protein of 22.5 kDa which is in agreement with the previously determined subunit M, of the purified enzyme. A putative 5-phosphoribosyl-a-1 -diphosphate (PRPP) binding site has been identified which is similar to the PRPP binding site of the yeast Uracil Phosphoribosyltransferase.
Majid Mehtali - One of the best experts on this subject based on the ideXlab platform.
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in vivo cancer gene therapy by adenovirus mediated transfer of a bifunctional yeast cytosine deaminase Uracil Phosphoribosyltransferase fusion gene
Cancer Research, 2000Co-Authors: Philippe Erbs, Etienne Regulier, Jacqueline Kintz, Pierre Leroy, Yves Poitevin, Francoise Exinger, Richard Jund, Majid MehtaliAbstract:Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase ( FCY1 ) and Uracil Phosphoribosyltransferase genes ( FUR1 ). This suicide gene, designated FCU1 , encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluoroUracil and 5-fluorouridine-5′-monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluoroUracil. Unexpectedly, although the Uracil Phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1 , its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1 . As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1 /5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.
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In Vivo Cancer Gene Therapy by Adenovirus-mediated Transfer of a Bifunctional Yeast Cytosine Deaminase/Uracil Phosphoribosyltransferase Fusion Gene
Cancer Research, 2000Co-Authors: Philippe Erbs, Etienne Regulier, Jacqueline Kintz, Pierre Leroy, Yves Poitevin, Francoise Exinger, Richard Jund, Majid MehtaliAbstract:Direct transfer of prodrug activation systems into tumors was demonstrated to be an attractive method for the selective in vivo elimination of tumor cells. However, most current suicide gene therapy strategies are still handicapped by a poor efficiency of in vivo gene transfer and a limited bystander cell killing effect. In this study, we describe a novel and highly potent suicide gene derived from the Saccharomyces cerevisiae cytosine deaminase ( FCY1 ) and Uracil Phosphoribosyltransferase genes ( FUR1 ). This suicide gene, designated FCU1 , encodes a bifunctional chimeric protein that combines the enzymatic activities of FCY1 and FUR1 and efficiently catalyzes the direct conversion of 5-FC, a nontoxic antifungal agent, into the toxic metabolites 5-fluoroUracil and 5-fluorouridine-5′-monophosphate, thus bypassing the natural resistance of certain human tumor cells to 5-fluoroUracil. Unexpectedly, although the Uracil Phosphoribosyltransferase activity of FCU1 was equivalent to that encoded by FUR1 , its cytosine deaminase activity was 100-fold higher than the one encoded by FCY1 . As a consequence, tumor cells transduced with an adenovirus expressing FCU1 (Ad-FCU1) were sensitive to concentrations of 5-FC 1000-fold lower than the ones used for cells transduced with a vector expressing FCY1 (Ad-FCY1). Furthermore, bystander cell killing was also more effective in cells transduced with Ad-FCU1 than in cultures infected with Ad-FCY1 or Ad-FUR1, alone or in combination. Finally, intratumoral injections of Ad-FCU1 into allo- or xenogeneic tumors implanted s.c. into mice, with concomitant systemic administration of 5-FC, led to substantial delays in tumor growth. These unique properties make of the FCU1 /5-FC prodrug activation system a novel and powerful candidate for cancer gene therapy strategies.
Glen N Barber - One of the best experts on this subject based on the ideXlab platform.
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the oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase Uracil Phosphoribosyltransferase suicide gene
Cancer Research, 2003Co-Authors: Mercedes Porosnicu, Abdul M Mian, Glen N BarberAbstract:Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/Uracil Phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluoroUracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-γ-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.
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The oncolytic effect of recombinant vesicular stomatitis virus is enhanced by expression of the fusion cytosine deaminase/Uracil Phosphoribosyltransferase suicide gene.
Cancer Research, 2003Co-Authors: Mercedes Porosnicu, Abdul M Mian, Glen N BarberAbstract:Vesicular stomatitis virus (VSV) has recently been demonstrated to exhibit significant oncolytic capabilities against a wide variety of tumor models in vitro and in vivo. To potentially enhance the oncolytic effect, we generated a novel recombinant VSV (rVSV) that expressed the fusion suicide gene Escherichia coli cytosine deaminase (CD)/Uracil Phosphoribosyltransferase (UPRT). rVSV encoding the CD/UPRT fusion gene (VSV-C:U) exhibited normal growth properties and generated high levels of biologically active CD/UPRT that could catalyze the modification of 5-fluorocytosine into chemotherapeutic 5-fluoroUracil (5-FU), which exhibited considerable bystander effect. Intratumoral inoculation of VSV-C:U in the presence of the systemically administered prodrug 5-fluorocytosine produced statistically significant reductions in the malignant growth of syngeneic lymphoma (A20) or mammary carcinoma (TSA) in BALB/c mice compared with rVSV treatments or with control 5-FU alone. Aside from detecting prolonged therapeutic levels of 5-FU in VSV-C:U-treated animals harboring TSA tumors and enhancing bystander killing of tumor cells, we demonstrated marked activation of IFN-γ-secreting cytotoxic T cells by enzyme-linked immunospot analysis that may have also facilitated tumor killing. In conclusion, the insertion of the fusion CD/UPRT suicide gene potentiates the oncolytic efficiency of VSV by generating a strong bystander effect and by contributing to the activation of the immune system against the tumor without detrimentally altering the kinetics of virus-mediated oncolysis and may be useful in the treatment of malignant disease.
