5 results on '"Heckl D"'
Search Results
2. Endogenous Tumor Suppressor microRNA-193b: Therapeutic and Prognostic Value in Acute Myeloid Leukemia.
- Author
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Bhayadia R, Krowiorz K, Haetscher N, Jammal R, Emmrich S, Obulkasim A, Fiedler J, Schwarzer A, Rouhi A, Heuser M, Wingert S, Bothur S, Döhner K, Mätzig T, Ng M, Reinhardt D, Döhner H, Zwaan CM, van den Heuvel Eibrink M, Heckl D, Fornerod M, Thum T, Humphries RK, Rieger MA, Kuchenbauer F, and Klusmann JH
- Subjects
- Animals, Cell Growth Processes genetics, Down-Regulation, Genes, Tumor Suppressor, Heterografts, Homeodomain Proteins genetics, Humans, Leukemia, Myeloid, Acute pathology, Leukemia, Myeloid, Acute therapy, MAP Kinase Signaling System, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, MicroRNAs genetics, Myeloid Ecotropic Viral Integration Site 1 Protein genetics, Prognosis, Leukemia, Myeloid, Acute genetics, MicroRNAs biosynthesis
- Abstract
Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.
- Published
- 2018
- Full Text
- View/download PDF
3. Pooled Generation of Lentiviral Tetracycline-Regulated microRNA Embedded Short Hairpin RNA Libraries.
- Author
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Adams FF, Hoffmann T, Zuber J, Heckl D, Schambach A, and Schwarzer A
- Subjects
- Cell Line, Humans, Gene Library, Genetic Vectors genetics, Lentivirus genetics, MicroRNAs genetics, RNA, Small Interfering genetics, Tetracycline
- Abstract
Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.
- Published
- 2018
- Full Text
- View/download PDF
4. An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.
- Author
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Adams FF, Heckl D, Hoffmann T, Talbot SR, Kloos A, Thol F, Heuser M, Zuber J, Schambach A, and Schwarzer A
- Subjects
- Cell Line, Doxycycline pharmacology, Gene Knockdown Techniques, Humans, Promoter Regions, Genetic, Cloning, Molecular, Genetic Vectors genetics, Lentivirus genetics, MicroRNAs genetics, RNA Interference, RNA, Small Interfering genetics
- Abstract
RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
5. MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy.
- Author
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Lachmann N, Jagielska J, Heckl D, Brennig S, Pfaff N, Maetzig T, Modlich U, Cantz T, Gentner B, Schambach A, and Moritz T
- Subjects
- Animals, Cell Line, Female, Male, Mice, Mice, Nude, Transgenes, B-Lymphocytes immunology, Down-Regulation, Gene Targeting adverse effects, Genetic Vectors, Hematopoiesis genetics, MicroRNAs genetics, T-Lymphocytes immunology
- Abstract
Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.
- Published
- 2012
- Full Text
- View/download PDF
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