5 results on '"Opalka, Bertram"'
Search Results
2. Reliable Generation of Stable High Titer Producer Cell Lines for Gene Therapy.
- Author
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Rattmann, Ina, Kleff, Veronika, Feldmann, Anja, Ludwig, Carsten, Sorg, Ursula Regina, Opalka, Bertram, Moritz, Thomas, and Flasshove, Michael
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CELL lines ,GENE therapy ,GENETIC transformation ,HEMATOPOIETIC stem cells ,HEMATOPOIESIS - Abstract
Objective: Retroviral vectors represent one of the most robust technologies for in vivo expression of heterologous gene sequences and are still the most commonly used vectors in clinical gene therapy trials. The production of high titer retroviral preparations, however, can be a problematic procedure for certain constructs. Methods: GALV- or RD114-pseudotyped retroviral particles carrying selectable fluorescence markers or drug resistance genes, such as the green fluorescent protein (GFP) or the O
6 -methylguanine-DNA-methyltransferase (MGMT) mutants, were used to stably transduce Phoenix-(FNX-)eco cells. Thereafter, a polyclonal population of producer cells was generated by enriching cells with high marker gene expression. In addition, single producer clones were selected by limiting dilution. Results: Retroviral titers were increased 1–2 logs by enriching for a polyclonal population of producer cells, and selection of single producer clones allowed another 1- to 2-log increase in titers. Using this method, reproducibly high titer viral preparations allowing efficient transduction of hematopoietic stem cells were generated. Conclusion: A reliable and time-effective method to generate stable high titer producer cells based on the FNX-cell line for problematic retroviral vector constructs is described. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]- Published
- 2007
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3. Apoptotic Genes in Cancer Therapy.
- Author
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Opalka, Bertram, Dickopp, Alexandra, and Kirch, Hans-Christoph
- Subjects
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APOPTOSIS , *GENE therapy , *CANCER treatment , *CELL death , *GENES - Abstract
Induction of apoptosis in malignant cells is a major goal of cancer therapy in general and of certain cancer gene therapy strategies in particular. Numerous apoptosis-regulating genes have been evaluated for this purpose. Besides the most prominent p53 gene others include p16, p21, p27, E2F genes, FHIT, PTEN and CASPASE genes. Recently, the potential for therapy of an adenoviral gene, E1A, known for a long time for its apoptosis-inducing activity, has been discovered. In experimental settings, these genes have proven their tumor-suppressive and apoptosis-inducing activity. Clinical trials are currently being performed with selected genes. By far the most studies transfer the p53 gene using retro- or adenoviral vectors. Disease stabilization or other benefits were observed in a limited number of patients when p53 was applied alone or in combination with cytotoxic drugs. A second proapoptotic gene that has entered clinical trials is adenovirus E1A. Here, too, disease stabilization as well as/or local regression in one case have been demonstrated in selected patients. In all cases, side effects were tolerable. To further improve E1A as a therapeutic transgene, we have deleted transforming domains from the adenovirus 5 and 12 13S cDNAs. Mutants were derived which had completely lost their transforming activity in combination with the E1B oncogene but retained a pronounced tumor-suppressive activity. Cells transduced with these constructs showed a highly reduced ability to grow in soft agar, and tumor growth in nude mice could be substantially suppressed. Outgrowing tumors had lost E1A expression when analyzed in Western blots. These E1A constructs may represent valuable tools for cancer gene therapy in the future.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]
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- 2002
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4. Gene Therapy of βc-Deficient Pulmonary Alveolar Proteinosis (βc-PAP): Studies in a Murine in vivo Model.
- Author
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Kleff, Veronika, Sorg, Ursula R, Bury, Carsten, Suzuki, Takuji, Rattmann, Ina, Jerabek-Willemsen, Moran, Poremba, Christopher, Flasshove, Michael, Opalka, Bertram, Trapnell, Bruce, Dirksen, Uta, and Moritz, Thomas
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GENE therapy , *PULMONARY alveoli , *GRANULOCYTE-macrophage colony-stimulating factor , *HEMATOPOIETIC stem cells , *BONE marrow , *MICROBIAL genetics , *IMMUNE system - Abstract
Pulmonary alveolar proteinosis (PAP) due to deficiency of the common β-chain (βc) of the interleukin–3 (IL–3)/IL–5/granulocyte–macrophage colony–stimulating factor (GM–CSF) receptors is a rare monogeneic disease characterized by functional insufficiency of pulmonary macrophages. Hematopoietic stem cell gene therapy for restoring expression of βc-protein in the hematopoietic system may offer a curative approach. Toward this end, we generated a retroviral construct expressing the murine βc (mβc) gene and conducted investigations in a murine model of βc-deficient PAP. Functional correction of mβc activity in mβc–/– bone marrow (BM) cells was demonstrated by restoration of in vitro colony formation in response to GM-CSF. In addition, in a murine in vivo model of mβc-deficient PAP mβc gene transfer to hematopoietic stem cells not only restored the GM-CSF-sensitivity of hematopoietic progenitor cells but also, within a period of 12 weeks, almost completely reversed the morphologic features of surfactant accumulation. These results were obtained despite modest transduction levels (10–20%) and, in comparison to wild-type mice, clearly reduced βc expression levels were detected in hematopoietic cells. Therefore, our data demonstrating genetic and functional correction of mβc–/– deficiency in vitro as well as in a murine in vivo model of PAP strongly suggest gene therapy as a potential new treatment modality in βc-deficient PAP.Molecular Therapy (2008); 16 4 757–764 doi:10.1038/mt.2008.7 [ABSTRACT FROM AUTHOR]
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- 2008
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5. O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: Studies addressing safety issues
- Author
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Sorg, Ursula R., Kleff, Veronika, Fanaei, Sepideh, Schumann, Alexandra, Moellmann, Michael, Opalka, Bertram, Thomale, Jürgen, and Moritz, Thomas
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METHYLTRANSFERASES , *GENE therapy , *G proteins , *ALKYLATING agents , *MUTAGENESIS , *HEMATOPOIETIC stem cells , *GENETIC toxicology - Abstract
Abstract: As haematopoietic stem cell gene therapy utilizing O6-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O6 alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level. [Copyright &y& Elsevier]
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- 2007
- Full Text
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