The Experts below are selected from a list of 18996 Experts worldwide ranked by ideXlab platform
Philippe Moullier - One of the best experts on this subject based on the ideXlab platform.
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Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates
PLoS ONE, 2014Co-Authors: Caroline Le Guiner, Marie Devaux, Yan Cherel, Philippe Moullier, Knut Stieger, Alice Toromanoff, Mickaël Guilbaud, Alexandra Mendes-madeira, Lydie Guigand, Fabienne RollingAbstract:Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Kru ̈ ppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric Transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive Transactivators with the potential to not trigger the host immune system are still needed.
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Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle
Molecular Therapy, 2004Co-Authors: Pierre Chenuaud, Hermann Bujard, Thibaut Larcher, Joseph E. Rabinowitz, Nathalie Provost, Béatrice Joussemet, Richard J.s. Samulski, Didier Favre, Philippe MoullierAbstract:Recombinant adeno-associated virus (rAAV) vector supports long-term transgene expression from skeletal muscle in most mammals, including human. In some instances, the requirement for tight control of the transgene expression is expected. The original tetracycline-dependent system using the rtTA (Dox-on) transactivator displayed a baseline activity in the off state but improved versions are now available and need to be evaluated in a single-rAAV-vector strategy. In the present study we cloned, in three different orientations, the two expression cassettes responsible for doxycycline-mediated transgene regulation and further evaluated the basal and inducible activity of the recently described rtTA2(S)-S2, rtTA2(S)-M2, and rtTA2(S)-M2n/s Transactivators. Evaluations were conducted in vivo in mice and nonhuman primates using the respective homologous erythropoietin cDNA as a reporter gene because of its sensitive detection by ELISA. The woodchuck hepatitis virus posttranscriptional regulatory element sequence was also introduced to enhance further the stringency with respect to basal activity in the absence of inducer.
Hermann Bujard - One of the best experts on this subject based on the ideXlab platform.
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Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle
Molecular Therapy, 2004Co-Authors: Pierre Chenuaud, Hermann Bujard, Thibaut Larcher, Joseph E. Rabinowitz, Nathalie Provost, Béatrice Joussemet, Richard J.s. Samulski, Didier Favre, Philippe MoullierAbstract:Recombinant adeno-associated virus (rAAV) vector supports long-term transgene expression from skeletal muscle in most mammals, including human. In some instances, the requirement for tight control of the transgene expression is expected. The original tetracycline-dependent system using the rtTA (Dox-on) transactivator displayed a baseline activity in the off state but improved versions are now available and need to be evaluated in a single-rAAV-vector strategy. In the present study we cloned, in three different orientations, the two expression cassettes responsible for doxycycline-mediated transgene regulation and further evaluated the basal and inducible activity of the recently described rtTA2(S)-S2, rtTA2(S)-M2, and rtTA2(S)-M2n/s Transactivators. Evaluations were conducted in vivo in mice and nonhuman primates using the respective homologous erythropoietin cDNA as a reporter gene because of its sensitive detection by ELISA. The woodchuck hepatitis virus posttranscriptional regulatory element sequence was also introduced to enhance further the stringency with respect to basal activity in the absence of inducer.
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Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo.
Gene Therapy, 2003Co-Authors: Jonna K. Koponen, Hermann Bujard, Wolfgang Hillen, Hanna M. Kankkonen, J Kannasto, T. Wirth, Seppo Ylä-herttualaAbstract:Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2 S -M2 shows a tight control of gene expression in vitro and in vivo
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exploring the sequence space for tetracycline dependent transcriptional activators novel mutations yield expanded range and sensitivity
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Stefanie Urlinger, Udo Baron, Marion Thellmann, Mazahir T Hasan, Hermann Bujard, Wolfgang HillenAbstract:Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of Ptet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2S-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting Transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.
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tetracycline controlled transcription in eukaryotes novel Transactivators with graded transactivation potential
Nucleic Acids Research, 1997Co-Authors: Udo Baron, Manfred Gossen, Hermann BujardAbstract:Several tetracycline-controlled Transactivators (tTA) were generated which differ in their activation potential by >3 orders of magnitude. The Transactivators are fusions between the Tet repressor and minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). By reducing the VP16 moiety of the previously described tTA to 12 amino acids, potential targets for interactions with various cellular transcription factors were eliminated, as were potential epitopes which may elicit a cellular immune response. When compared with the originally described tTA, these new Transactivators are tolerated at higher intracellular concentrations. This will facilitate establishment of tet regulatory systems under a variety of conditions, but particularly when cell type-restricted tetracycline-controlled gene expression is to be achieved in transgenic organisms via homologous recombination.
Fabienne Rolling - One of the best experts on this subject based on the ideXlab platform.
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Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in
2016Co-Authors: Nonhuman Primates, Caroline Le Guiner, Marie Devaux, Lydie Guig, Yan Cherel, Fabienne RollingAbstract:Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric Transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive Transactivators with the potential to not trigger the host immun
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Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates
PLoS ONE, 2014Co-Authors: Caroline Le Guiner, Marie Devaux, Yan Cherel, Philippe Moullier, Knut Stieger, Alice Toromanoff, Mickaël Guilbaud, Alexandra Mendes-madeira, Lydie Guigand, Fabienne RollingAbstract:Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Kru ̈ ppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric Transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive Transactivators with the potential to not trigger the host immune system are still needed.
David G Schatz - One of the best experts on this subject based on the ideXlab platform.
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a modified tetracycline regulated system provides autoregulatory inducible gene expression in cultured cells and transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Penny E Shockett, Michael J Difilippantonio, Nathan Hellman, David G SchatzAbstract:Abstract A system for tetracycline-regulated inducible gene expression was described recently which relies on constitutive expression of a tetracycline-controlled transactivator (tTA) fusion protein combining the tetracycline repressor and the transcriptional activation domain of VP16 [Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551]. This system yielded only low levels of transactivator protein, probably because tTA is toxic. To avoid this difficulty, we placed the tTA gene under the control of the inducible promoter to which tTA binds, making expression of tTA itself inducible and autoregulatory. When used to drive expression of the recombination activating genes 1 and 2 (RAG-1 and RAG-2), the autoregulatory system yielded both substantially higher levels of variable (diversity) joining [V(D)J] recombination activity (70-fold on average) and inducible expression in a much larger fraction of transfected cells (autoregulatory, 90%, vs. constitutive, 18%). In addition, this system allowed the creation of transgenic mice in which expression of a luciferase transgene was inducible tens to hundreds of times the basal levels in most tissues examined. Induced levels of expression were highest in thymus and lung and appear to be substantially higher than in previously reported inducible luciferase transgenic mice created with the constitutive system. With the modified system, inducible transactivator mRNA and protein were easily detected in cell lines by RNA and Western blotting, and transactivator mRNA was detected by RNA blotting in some tissues of transgenic mice. This autoregulatory system represents an improved strategy for tetracycline-regulated gene expression both in cultured cells and in transgenic animals.
Caroline Le Guiner - One of the best experts on this subject based on the ideXlab platform.
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Transgene Regulation Using the Tetracycline-Inducible TetR-KRAB System after AAV-Mediated Gene Transfer in
2016Co-Authors: Nonhuman Primates, Caroline Le Guiner, Marie Devaux, Lydie Guig, Yan Cherel, Fabienne RollingAbstract:Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric Transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive Transactivators with the potential to not trigger the host immun
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Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates
PLoS ONE, 2014Co-Authors: Caroline Le Guiner, Marie Devaux, Yan Cherel, Philippe Moullier, Knut Stieger, Alice Toromanoff, Mickaël Guilbaud, Alexandra Mendes-madeira, Lydie Guigand, Fabienne RollingAbstract:Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Kru ̈ ppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric Transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive Transactivators with the potential to not trigger the host immune system are still needed.
