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  • 1
    Keywords: CANCER ; CELLS ; EXPRESSION ; tumor ; TUMOR-CELLS ; CELL ; Germany ; KINASE ; THERAPY ; GENE ; GENE-EXPRESSION ; PROTEIN ; cell line ; TUMORS ; LINES ; DNA ; INFECTION ; REDUCTION ; CELL-LINES ; PHOSPHORYLATION ; treatment ; PARTICLES ; virus ; ISOFORM ; gene expression ; PROMOTER ; DECREASE ; ELEMENTS ; NUMBER ; CELL-LINE ; LINE ; TRANSFORMATION ; GLUCOSE ; FLUORESCENCE ; REGULATORY ELEMENTS ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; VIRUS THYMIDINE KINASE ; TUMOR-CELL-LINES ; HIGH-LEVEL ; HaCaT ; Ras ; THYMIDINE KINASE ; SRC ONCOGENE ; ADENOASSOCIATED VIRUS VECTORS ; CYSTIC-FIBROSIS ; glucose transporter promoter,HSV thymidine kinase,suicide gene,AAV
    Abstract: In order to achieve tumor-specific targeting of adeno-associated virus (AAV)-mediated gene expression, the promoter of the glucose transporter isoform 1 (GLUT1) gene was cloned upstream of the enhanced green fluorescence protein (EGFP) and the herpes simplex virus thymidine kinase (HSVtk) gene. FACS analysis performed at 48 h after transient infection with rAAV/cytomegalovirus (CMV)egfp viral particles revealed an increase of fluorescence in all the cell lines tested. However, EGFP expression under control of the GLUT1 promoter element (rAAV/GTI-1.3egfp) was limited to the tumor cells and oncogene-transformed cells. Evidence for phosphorylation of the HSVtk substrates ganciclovir (GCV) and I-125-deoxycytidine was found in all transfected tumor cell lines compared to noninfected controls (HCT116: 111%; MH3924A: 130%; HaCaT-RT3: 257% increase), but not in HaCaT and HUVEC cells. Furthermore, tumor cells and the oncogene-transformed (ras) cell line HaCaT-RT3 showed a GCV-induced reduction in cell number (HCT116:-71%; MH3924A:-43% and HaCaT-RT3:-31%). No statistically relevant cytotoxic effect was observed in HaCaT (6% decrease) and HUVEC cells (2% decrease). Furthermore, a reduction of H-3-thymidine incorporation into the DNA was seen after treatment with GCV (HCT116: 38%; MH3924A: 33% and HaCaT-RT3: 37% decrease). In a therapy study of HSVtk-expressing tumors with GCV, we achieved total tumor remission
    Type of Publication: Journal article published
    PubMed ID: 14681725
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  • 2
    Keywords: APOPTOSIS ; CANCER ; CELLS ; EXPRESSION ; GROWTH ; IN-VITRO ; tumor ; TUMOR-CELLS ; CELL ; Germany ; human ; KINASE ; MODEL ; MODELS ; THERAPY ; VITRO ; EXPOSURE ; GENE ; PROTEIN ; EFFICIENCY ; cell line ; TISSUE ; TUMORS ; gene therapy ; LINES ; MICE ; TRANSDUCTION ; MR ; CELL-LINES ; treatment ; SUSCEPTIBILITY ; NOD/Scid mice ; virus ; VECTORS ; PROMOTER ; IMMUNODEFICIENT MICE ; CELL-LINE ; chemotherapy ; FUSION ; LINE ; CANCER-CELLS ; STRATEGIES ; GENE-THERAPY ; RECOMBINANT ADENOASSOCIATED VIRUS ; SARCOMA ; GREEN FLUORESCENT PROTEIN ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; THYMIDINE KINASE ; adeno-associated virus 2 ; GANCICLOVIR ; suicide gene therapy ; TRANSGENE EXPRESSION
    Abstract: Soft-tissue sarcomas are mesenchymal tumors that respond poorly to systemic chemotherapy. Suicide gene therapy may be an alternative treatment strategy. Here we show a high susceptibility of human sarcoma cell lines for recombinant adeno-associated virus 2 (rAAV-2) suicide vectors: connective tissue sarcoma (HS-1), fibrosarcoma (HT-1080), Ewing sarcoma (RD-ES), Askin tumor (SK-N-MC), rhabdomyosarcoma (A-204) and soft-tissue sarcoma (WSKL-1). Several vectors containing the thymidine kinase (TK) gene under the control of either the cytomegalovirus promoter or the elongation-factor 1 alpha (EF1alpha) promoter were cloned and tested. Higher expression levels of the transgene were observed in the sarcoma lines when using the EF1alpha-suicide gene-containing vectors. A complete eradication of rAAV-2-EF1alpha-TK/eGFP (TK/enhanced green fluorescent protein fusion gene)-transduced tumor cells was shown following exposure to ganciclovir (2.5 mug/ml) in vitro, while at this dose level 〉 90% of mock-transduced tumor cells survived. Xenotransplantation tumor models ( intraperitoneal, subcutaneous) for the human sarcoma cell line HS-1 were established in nonobese diabetic/severe-combined immunodeficient mice. Mice transplanted with rAAV-2-EF1alpha-TK/eGFP-transduced and ganciclovir-exposed tumor cells survived 〉5 months while in the nontransduced group all mice had died approximately 1 month after inoculation. These data hold promise for further development of rAAV-2-based suicide gene therapy of sarcomas
    Type of Publication: Journal article published
    PubMed ID: 15280909
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  • 3
    Abstract: Autonomous parvoviruses possess an intrinsic oncotropism based on viral genetic elements controlling gene expression and genome replication. We constructed a hybrid vector consisting of the H1 parvovirus-derived expression cassette comprising the p4 promoter, the ns1 gene and the p38 promoter flanked by the adeno-associated viruses 2 ( AAV2) inverted terminal repeats and packaged into AAV2 capsids. Gene transduction using this vector could be stimulated by coinfection with adenovirus, by irradiation or treatment with genotoxic agents, similar to standard AAV2 vectors. However, the latter were in most cases less efficient in gene transduction than the hybrid vector. With the new vector, tumor cell-selective increase in transgene expression was observed in pairs of transformed and non-transformed cells, leading to selective killing of the transformed cells after expression of a prodrug-converting enzyme. Preferential gene expression in tumor versus normal liver tissue was also observed in vivo in a syngeneic rat model. Comparative transduction of a panel of different tumor cell lines with the H1 and the H1/AAV hybrid vector showed a preference of each vector for distinct cell types, probably reflecting the dependence of the viral tropism on capsid determinants
    Type of Publication: Journal article published
    PubMed ID: 18202715
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  • 4
    Keywords: CANCER ; CELLS ; EXPRESSION ; GROWTH ; CELL ; SYSTEM ; transcription ; MICE ; TRANSDUCTION ; DNA ; MINUTE VIRUS ; REPLICATION ; ADENOVIRUS ; AUTONOMOUS PARVOVIRUSES ; RECOMBINANT PARVOVIRUS ; ADENOASSOCIATED VIRUS VECTORS ; pXX6 ; virus helper ; virus production
    Abstract: Preclinical studies using various cell culture and animal systems highlight the potential of recombinant rodent parvoviruses (recPVs) for cancer therapy. Production of these viruses is, however, not efficient and this hampers the clinical applications of these agents. In this study, we show that the adenovirus genes E2a, E4(orf6) and VA RNA increase the production of recPVs by more than 10-fold and reduce the time of production from 3 to 2 days in HEK293T cells. The helper effects of these genes can be observed with different recPVs, regardless of the nature and size of the inserted transgene. Furthermore, we generated a recombinant Adenovirus 5 carrying the parvovirus VP transcription unit. This helper, named Ad-VP, allows recPVs to be efficiently produced through a protocol based only on cell infection, making possible to use cell lines, such as NB324K, which are good producers of parvoviruses but are hardly transfectable. Hence, we could further improve viral titers and reduce time and costs of production. This Ad-VP helper-based protocol could be scaled up to a bioreactor format for the generation of the large amounts of recPVs needed for future clinical applications.
    Type of Publication: Journal article published
    PubMed ID: 21102423
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  • 5
    Keywords: EXPRESSION ; IN-VITRO ; HEPATOCELLULAR-CARCINOMA ; PROSTATE-CANCER ; GENE-THERAPY ; pancreatic cancer ; SERIAL ANALYSIS ; oncolysis ; STEM-CELL ANTIGEN ; SINGLE-CHAIN ANTIBODY ; ONCOLYTIC VIRUSES ; DEOXYNUCLEOSIDE ANALOGS ; FLUDARABINE PHOSPHATE ; measles virus ; prodrug-converting enzyme ; PURINE NUCLEOSIDE PHOSPHORYLASE
    Abstract: No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high-and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.
    Type of Publication: Journal article published
    PubMed ID: 21701532
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  • 6
    Keywords: CELLS ; EXPRESSION ; carcinoma ; PROTEINS ; mechanisms ; BREAST-CANCER ; PROGRESSION ; statistics ; oligonucleotides ; INTEGRIN ; heredity ; MANAGEMENT ; TUMOR-METASTASIS ; CD44 ; antisense-oligonucleotide ; CC531 colorectal cancer cells ; extracellular matrix protein:osteopontin ; liver metastasis of colorectal cancer ; modulation of expression
    Abstract: Development of hepatic metastasis is responsible for most of colorectal cancer-related deaths. Osteopontin (OPN) is a small integrin-binding N-linked glycoprotein, which plays a crucial role in the formation of hepatic metastasis. This study aimed to suppress Opn expression by an antisense-oligonucleotide (ASO(Opn)) to decrease liver metastasis in vivo. The effect of ASO(Opn) was investigated in vitro in CC531(lacZ) colorectal cancer cells in comparison to sense (SO) or nonsense (NSO) oligomers, by determining mRNA and protein expression levels, as well as cell survival. For in vivo treatment, CC531(lacZ) cells were intraportally inoculated into rats to compare the effects of ASO, SO and NSO oligomers, following prolonged subcutaneous administration by osmotic mini-pumps. The resulting CC531(lacZ) tumor cell load in the liver was measured by a beta-galactosidase assay. Proliferation of CC531(lacZ) cells in vitro was significantly decreased after ASO(Opn) and SO treatment (P〈0.001). Liver metastasis development was reduced as long as ASO(Opn) was administered, but this effect was rapidly blunted following the end of the ASO(Opn) administration. In contrast, administration of the SO resulted in a tumor load reduction, which surprisingly surpassed the ASO(Opn) effect in vivo in terms of a long-lasting metastasis suppression, which was accompanied with increased survival of the animals. Administration of the ASO(Opn) in rats was effective in decreasing their liver metastasis. The short-lived effect might be extended by modifications suited to increase the ASOs' half-life. In addition, there was a superior anti-metastatic effect caused by the SO, which has not been reported previously.
    Type of Publication: Journal article published
    PubMed ID: 21852811
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  • 7
    Keywords: CANCER ; CANCER CELLS ; CELLS ; INHIBITION ; PATHWAY ; TOXICITY ; SITE ; SITES ; GENE ; GENES ; PROTEIN ; PROTEINS ; transcription ; MICE ; ACTIVATION ; DNA ; INFECTION ; FAMILY ; TRANSCRIPTION FACTOR ; colon ; BINDING ; CYCLE ; virus ; TRANSCRIPTION FACTORS ; PROMOTER ; NUMBER ; CAPSID PROTEIN ; CANCER-CELLS ; COLON-CANCER ; MUTATIONS ; MINUTE VIRUS ; REPLICATION ; beta-catenin ; CONSTITUTIVE ACTIVATION ; autonomous parvovirus ; WNT SIGNALING PATHWAY ; signaling ; colon cancer ; INCREASE ; cancer therapy ; HUMAN-FIBROBLASTS ; oncolytic virus ; VIRUS-INFECTION ; H-1 PARVOVIRUS ; function ; Wnt signaling ; H1 ; SIMIAN VIRUS-40
    Abstract: The Wnt signaling pathway is activated by mutations in the adenomatous polyposis coli (APC) or beta-catenin genes in most colon cancers, leading to the transactivation of promoters containing binding sites for the Tcf/LEF family of transcription factors. We have previously shown that it is possible to confer colon cancer specificity on autonomous parvoviruses by inserting Tcf sites into the viral P4 promoter. The mutant Tcf promoters were responsive to activation of the Wnt pathway but the viruses replicated poorly. We show here that reduction of the number of Tcf sites from four to two leads to an increase in the efficiency of replication and toxicity of the viruses in Co115 colon cancer cells, with only a small reduction in selectivity for cells with an active Wnt signaling pathway. Despite this improvement, virus production by most colon cancer cells remained low. Analysis of parental phH1 virus infection of SW480 colon cancer cells showed that the nonstructural and capsid proteins were expressed, but single stranded DNA and progeny virus were not produced. This defect reflects the dependence of autonomous parvoviruses on host functions for many steps in their replication cycle and represents a major limitation to the use of selectively replicating parvoviruses for colon cancer therapy
    Type of Publication: Journal article published
    PubMed ID: 16151476
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  • 8
    Keywords: tumor ; PHASE-I ; ANTIGEN ; OVARIAN-CANCER ; PROSTATE-CANCER ; FUSION ; GENE-THERAPY ; ADENOVIRUS ; COLONY-STIMULATING FACTOR ; CYTOSINE DEAMINASE ; SODIUM-IODIDE SYMPORTER ; CELL CARCINOMA ; HNSCC ; EGFR ; VIROTHERAPY ; cetuximab ; oncolytic measles virus ; prodrug converting enzyme
    Abstract: First-line treatment of recurrent and/or refractory head and neck squamous cell carcinoma (HNSCC) is based on platinum, 5-fluorouracil (5-FU) and the monoclonal anti EGFR antibody cetuximab. However, in most cases this chemoimmunotherapy does not cure the disease, and more than 50% of HNSCC patients are dying because of local recurrence of the tumors. In the majority of cases, HNSCC overexpress the epidermal growth factor receptor (EGFR), and its presence is associated with a poor outcome. In this study, we engineered an EGFR-targeted oncolytic measles virus (MV), armed with the bifunctional enzyme cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT). CD/UPRT converts 5-fluorocytosine (5-FC) into the chemotherapeutic 5-FU, a mainstay of HNSCC chemotherapy. This virus efficiently replicates in and lyses primary HNSCC cells in vitro. Arming with CD/UPRT mediates efficient prodrug activation with high bystander killing of non-infected tumor cells. In mice bearing primary HNSCC xenografts, intratumoral administration of MV-antiEGFR resulted in statistically significant tumor growth delay and prolongation of survival. Importantly, combination with 5-FC is superior to virus-only treatment leading to significant tumor growth inhibition. Thus, chemovirotherapy with EGFR-targeted and CD/UPRT-armed MV is highly efficacious in preclinical settings with direct translational implications for a planned Phase I clinical trial of MV for locoregional treatment of HNSCC.
    Type of Publication: Journal article published
    PubMed ID: 22076043
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  • 9
    Keywords: CANCER ; liver ; MESSENGER-RNA ; ENTRY ; adenocarcinoma ; REPLICATION ; GLYCOPROTEINS ; VIROTHERAPY ; CHEMOVIROTHERAPY
    Abstract: Precise oncotropism is required for successful systemic administration of next-generation oncolytic measles viruses (MVs). We have previously established a system for efficient post-entry targeting by insertion of synthetic microRNA target sites (miRTS) into the MV genome, thereby repressing replication in the presence of cognate microRNAs. Thus, differential expression of microRNAs, as frequently observed in normal compared with malignant tissues, can be exploited to increase vector specificity and safety. Here we report the combination of miRTS for different microRNAs in a single vector to detarget pivotal organs at risk during systemic administration (liver, brain, gastrointestinal tract). Accordingly, miRTS for miR-122, miR-7 and miR-148a that are enriched in these tissues were inserted to create multi-tissue-detargeted MV (MV-EGFP(mtd)). Replication of MV-EGFP(mtd) is repressed in cell lines as well as in non-transformed primary human hepatocytes and liver slices expressing cognate microRNAs. Oncolytic potency of MV-EGFP(mtd) is retained in a model of pancreatic cancer in vitro and in vivo. This work is a proof-of-concept that favorable expression profiles of multiple microRNAs can be exploited concomitantly to reshape the tropism of MV without compromising oncolytic efficacy. This strategy can be adapted to different vectors and cancer entities for safe and efficient high-dose systemic administration in clinical trials.
    Type of Publication: Journal article published
    PubMed ID: 25145311
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  • 10
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; IN-VITRO ; TUMOR-CELLS ; carcinoma ; Germany ; MICROSCOPY ; THERAPY ; VITRO ; DISEASE ; GENE ; GENES ; PROTEIN ; LINES ; gene transfer ; GENE-TRANSFER ; INFECTION ; ANTIGEN ; BINDING ; CELL-LINES ; MOLECULE ; antibodies ; antibody ; virus ; leukemia ; LINE ; DELIVERY ; VACCINE ; FLUORESCENCE ; cell lines ; ADENOVIRUS ; FUSION PROTEIN ; LEUKEMIA-CELLS ; targeting ; RE ; targeted ; NEWCASTLE-DISEASE-VIRUS ; SINGLE-CHAIN ANTIBODY ; bispecific ; CLONED CDNA ; enhanced green fluorescent protein ; FOREIGN GENE ; Newcastle disease virus ; RNA VIRUSES
    Abstract: We developed a novel strategy to target recombinant Newcastle disease virus (NDV) to tumor cells for gene therapy. Modifying the virus with a bispecific fusion protein allowed virus receptor-independent tumor cell binding and gene transfer. The targeting molecule alphaHN-IL-2 contains an scFv antibody cloned from a neutralizing hemagglutinin-neuraminidase (HN)-specific hybridoma linked to the human cytokine IL-2. A recombinant NDV expressing the enhanced green fluorescent protein (NDFL-EGFP) was applied to show the expression of foreign genes in virus-infected tumor cells. At 24 hours after infection with the modified virus (NDFL-EGFP/alphaHN-IL-2), FACS analysis and fluorescence microscopy revealed neutralization of natural infection in IL-2 receptor-negative Jurkat leukemia cells, but targeted expression of EGFP in IL-2 receptor-positive human leukemia-derived MT-2 cells. The targeted gene delivery of NDFL-EGFP/alphaHN-IL-2 in MT-2 cells was blocked by the target ligand human IL-2. Selective virus entry to IL-2 receptor bearing tumor cells was also observed in a mixture of Jurkat and MT-2 cell lines. These results demonstrate that a recombinant NDV carrying a foreign gene can be successfully targeted to a specific tumor through a bispecific protein, which thereby increases the selectivity of gene transfer
    Type of Publication: Journal article published
    PubMed ID: 15605075
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