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    Keywords: EXPRESSION ; Germany ; QUANTIFICATION ; TIME ; PATIENT ; treatment ; ALPHA ; ASSAY ; DECREASE ; POLYMERASE-CHAIN-REACTION ; INTERFERON
    Abstract: We developed a real-time quantitative polymerase chain reaction-based assay for quantification of PRV-1 mRNA. We found that the expression of PRV-1 in granulocytes of patients with polycythemia vera (PV) who were pretreated with phlebotomy or hydroxyurea was significantly higher than that in normal controls. Surprisingly, in PV patients who had received interferon-alpha (IFN) for five or more months no significant PRV-1 upregulation was found. Observation of four PV patients treated with IFN over six months revealed a uniform time- dependent decrease of initially upregulated PRV-1
    Type of Publication: Journal article published
    PubMed ID: 12651277
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  • 3
    Keywords: CELLS ; EXPRESSION ; BLOOD ; CELL ; Germany ; human ; KINASE ; THERAPY ; COMMON ; SITE ; SITES ; GENE ; GENE-EXPRESSION ; GENES ; GENOME ; PROTEIN ; PROTEINS ; transcription ; gene therapy ; LINES ; MICE ; TIME ; TRANSDUCTION ; PATIENT ; ACTIVATION ; TRANSCRIPTION FACTOR ; cell cycle ; CELL-CYCLE ; CYCLE ; FREQUENCY ; FREQUENCIES ; bone marrow ; BONE-MARROW ; MOUSE ; TRANSCRIPTION FACTORS ; gene expression ; VECTORS ; VECTOR ; IMMUNODEFICIENT MICE ; PCR ; HUMAN GENOME ; REGION ; REGIONS ; PROGENITOR CELLS ; SAFETY ; SELECTION ; HEMATOPOIETIC-CELLS ; PERIPHERAL-BLOOD ; GENE-THERAPY ; INTEGRATION SITE ; RETROVIRAL VECTORS ; peripheral blood progenitor cells ; ORIENTATION ; insertional mutagenesis ; N-MYC ; signaling ; INTEGRATION ; PROGRAM ; DETERMINANTS ; INTERFERENCE ; THERAPIES ; MYELOID-LEUKEMIA ; BLOOD PROGENITOR CELLS ; CPG ISLANDS ; favored integration ; ligation-mediated PCR ; NOD/SCID mouse assay ; retroviral vector transduction ; ZINC-FINGER PROTEIN
    Abstract: Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC
    Type of Publication: Journal article published
    PubMed ID: 15509505
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  • 4
    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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  • 5
    Keywords: CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; IONIZING-RADIATION ; IRRADIATION ; PROTECTION ; radiotherapy ; tumor ; TUMOR-CELLS ; carcinoma ; CELL ; Germany ; human ; LUNG ; THERAPY ; SYSTEM ; SYSTEMS ; GENE ; GENES ; PROTEIN ; TISSUE ; TRANSDUCTION ; INDEX ; TISSUES ; MR ; SUSCEPTIBILITY ; virus ; NO ; VECTORS ; ASSAY ; resistance ; VECTOR ; DAMAGE ; CANCER-CELLS ; CARCINOMA-CELLS ; LOCALIZATION ; SAFETY ; NORMAL TISSUE ; OVEREXPRESSION ; mutagenesis ; RECOMBINANT ADENOASSOCIATED VIRUS ; adeno-associated virus ; ADENOASSOCIATED VIRUS ; TUMOR CELLS ; RECOMBINANT ; adeno-associated virus 2 ; cervical carcinoma cells ; CANDIDATE GENES ; SUPEROXIDE-DISMUTASE ; HIGH-TITER ; INTRATRACHEAL INJECTION ; MNSOD-PL
    Abstract: Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although 〉 70% of all cells expressed SOD and significant amounts Of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection. (C) 2004 Elsevier Ireland Ltd, All rights reserved
    Type of Publication: Journal article published
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  • 6
    Keywords: CANCER ; IN-VITRO ; INVASION ; tumor ; CELL ; CELL LUNG-CANCER ; Germany ; IN-VIVO ; VITRO ; VIVO ; SYSTEM ; DISEASE ; MECHANISM ; prognosis ; mechanisms ; BIOLOGY ; cell cycle ; CELL-CYCLE ; CYCLE ; BREAST-CANCER ; PROGRESSION ; TUMOR PROGRESSION ; METASTASIS ; COLORECTAL-CANCER ; CARCINOMA-CELLS ; UROKINASE RECEPTOR ; PROGNOSTIC VALUE ; TRANSCRIPTIONAL REGULATION ; MINIMAL RESIDUAL DISEASE ; RE ; RESIDUAL DISEASE ; development ; TUMOR-CELL ; CANCERS ; HUMAN-COLON CANCER ; in vivo ; PLASMINOGEN-ACTIVATOR RECEPTOR ; RELEVANCE ; urokinase-receptor ; u-PAR ; INHIBITOR TYPE-1 ; UPA ; BONE-MARROW MICROMETASTASES ; dormancy ; LOCALIZED PROSTATE-CANCER
    Abstract: The relevance of the u-PA system in mediating tumor-associated proteolysis, invasion and metastasis, amongst other phenomena associated with tumor progression, has been clearly demonstrated in diverse cancer entities. This review will update on the biological and clinical relevance of the urokinase-receptor (u-PAR). Specifically, the article focuses on the potential importance of u-PAR for the development of minimal residual disease in solid cancer, and in this context reviews the biological relevance of the u-PAR for tumor cell dormancy. Furthermore, transcriptional mechanisms regulating u-PAR in vitro and in vivo, and their potential clinical and therapeutic relevance in gastrointestinal cancers, are elucidated
    Type of Publication: Journal article published
    PubMed ID: 16931909
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  • 7
    Keywords: SPECTRA ; CELLS ; BLOOD ; CELL ; Germany ; human ; THERAPY ; POPULATION ; SITE ; SITES ; GENE ; GENES ; GENOME ; PROTEIN ; transcription ; METABOLISM ; MICE ; TRANSDUCTION ; TRANSPLANTATION ; SEQUENCE ; VECTOR ; HUMAN GENOME ; REGION ; REGIONS ; STEM-CELLS ; HEMATOPOIETIC-CELLS ; mutagenesis ; insertional mutagenesis ; cord blood ; INTEGRATION ; PROGRAM ; RE ; LENTIVIRAL VECTOR ; ligation-mediated PCR ; SEVERE COMBINED IMMUNODEFICIENCY ; technique ; function ; hematopoietic stem cell ; REPOPULATING CELLS ; RELEVANCE ; progenitor cell ; HUMAN-GENOME ; CORD ; TRANSCRIPTION START REGIONS ; BLOOD-GROUP ; cord blood progenitors cells ; I-HYPERSENSITIVE SITES ; lentiviral vector transduction ; RETROVIRAL GENE MARKING ; scid repopulating cell
    Abstract: Background Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors. Methods We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into.NOD/SCID mice using a self-inactivating HIV-1-derived vector expressing the enhanced green fluorescent protein (EGFP). Results The mean of transduction of CD34(+) CB cells was 41%, as deduced from the percentage of EGFP(+) cells before transplantation. At 3 weeks post-transplantation, the average of EGFP+ cells in the human cell population was 65 +/- 8%, and increased to 75 +/- 10% at 12 weeks post-transplantation. In order to determine the proviral integration sites in human NOD/SCID repopulating cells (SRCs) we used the ligation-mediated polymerase chain I reaction (LM-PCR) technique. Sixty-eight percent of the integrations were found to be located in RefSeq genes, most of them in intron regions. Twenty percent of these integrations occurred within a distance of 10 kb from the transcription start site; a percentage that is significantly lower compared to that observed in cells transduced by gammaretroviral vectors. Sixty-two percent of integrations occurred in genes with a biological function in cell metabolism, and four integrations were located in genes with a role in turnorigenesis. Conclusions These investigations indicate that integration of lentiviral vectors in human repopulating cells capable of engrafting NOD/SCID mice preferentially occur in coding regions of the human genome. Nevertheless, the clustering of integrations at the transcriptional start is not as high as that observed for gammaretroviral vectors. Copyright (c) 2006 John Wiley & Sons, Ltd
    Type of Publication: Journal article published
    PubMed ID: 16960916
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  • 8
    Keywords: CELLS ; IN-VITRO ; SURVIVAL ; tumor ; CELL ; Germany ; human ; IN-VIVO ; MODEL ; MODELS ; THERAPY ; VITRO ; SYSTEM ; SYSTEMS ; GENE ; TUMORS ; gene therapy ; TIME ; PATIENT ; SUSCEPTIBILITY ; virus ; MOUSE ; IN-SITU ; VECTORS ; DIFFERENCE ; VECTOR ; DELETIONS ; FISH ; MOUSE MODEL ; GENE-THERAPY ; adeno-associated virus ; mesothelioma ; PLEURAL MESOTHELIOMA ; ONCOLOGY ; RECOMBINANT ; malignant pleural mesothelioma ; suicide gene therapy ; THERAPIES ; TUMORIGENICITY ; methods ; BONE ; MPM ; VECTOR STOCKS
    Abstract: Background: The median survival time of patients with malignant pleural mesothelioma (MPM) remains poor. Therefore, novel therapeutic options are in high demand, and well characterized model systems for in vitro/vivo screening have to be established. Material and Methods: For this purpose, 3 MPM cell lines (H-Meso-1, MSTO-211H, and NCI-H28) were characterized and tested for susceptibility to recombinant adeno-associated virus 2 (rAAV2)-based vectors which have the potential for a loco-regional application. Results: Using multiplex fluorescence in situ hybridization, several recurrent chromosomal aberrations were observed for each of the MPM cell lines. Tumorigenicity of H-Meso-1 and MSTO-211H cells was shown in an intraperitoneal NOD/SCID mouse model, whereas NCI-H28 cells did not yield any tumors. Although all 3 cell lines were readily susceptible to rAAV2 vectors, differences in susceptibility were observed (H-Meso-1 〉 NCI-H28 〉 MSTO-211H). Furthermore, the efficacy of a potential suicide gene therapy using an rAAV2 suicide vector-transduced MPM cell line was determined in a proof-of-feasibility in vivo experiment. Conclusion: The characterized cell lines described here may serve as a model for in vitro and in vivo preclinical gene therapy for the treatment of MPM using rAAV2 suicide vectors
    Type of Publication: Journal article published
    PubMed ID: 18322411
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  • 9
    Keywords: EXPRESSION ; IN-VIVO ; THERAPY ; TYROSINE KINASE ; GENE ; TRANSDUCTION ; resistance ; CHRONIC MYELOID-LEUKEMIA ; AAV ; VIRUS VECTORS ; IMATINIB STI571
    Abstract: Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1--ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded self-complementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations. On human CML cell lines, ssAAV2/2 and ssAAV2/6 were most efficient. On primary cells, ssAAV2/6 proved significantly more efficient (4.1 aEuroS +/-+/- aEuroS2.5%% GFP〈SU++〈/SU cells, p aEuroS== aEuroS0.011) than the other vectors (〈 1%%). The transduction efficiency could be significantly increased (45.5 aEuroS +/-+/- aEuroS13.4%%) by using dsAAV2/6 vectors (p aEuroS 〈 aEuroS0.001 vs. ssAAV2/6). In these settings, our data suggest conversion of single- to double-stranded DNA and cell binding/entry as rate-limiting steps. Furthermore, gene transfer was observed in both late and earlier CML (progenitor) populations. For the first time, efficient AAV gene transfer into human CML cells could be shown, with the potential for future clinical application.
    Type of Publication: Journal article published
    PubMed ID: 21323526
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  • 10
    Abstract: Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification-mediated PCR (LAM-PCR) or nonrestrictive LAM-PCR (nrLAM-PCR), both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.
    Type of Publication: Journal article published
    PubMed ID: 26052530
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