Inducible Transgenic Mouse Model Of RNA Toxicity
Mani S Mahadevan, Assistant Professor
Pathologyuniversity Of Virginia Charlottesville
Grant 5R01AR052771-04 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SMEP
Abstract: Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults. There are two types, DM1 and DM2, both being autosomal dominant disorders caused by expansions of microsatellite repeats within non-coding regions of their respective genes. DM1 is far more common; however both forms of DM are likely to share similar pathogenic mechanisms. The DM1 mutation is an expansion of a CTG triplet repeat in the 3´ untranslated region (3´UTR) of the DM protein kinase (DMPK) gene. A prevailing hypothesis in the field is that many aspects of DM are caused by the expression of the mutant mRNA. DM1 and DM2 represent the first examples of toxic RNA mediated disease pathogenesis. We have already developed and characterized extensively, a myoblast cell culture model to clearly demonstrate the toxic effects of the mutant DMPK mRNA on muscle differentiation. To study the hypothesis further, the aims of this proposal are to develop and characterize an inducible transgenic mouse model of RNA toxicity for DM type 1 (DM1) and to develop a siRNA (small interfering RNA) therapeutic approach to get rid of the toxic RNA which can be tested in both our cell culture and transgenic animal models. The development of transgenic mouse models will aid in understanding disease pathogenesis and will also provide a system with which to test out potential therapeutic .strategies. The ability to control gene expression through an inducible system will enable better characterization of and better correlation with the onset and levels of expression of the toxic RNA in DM1 and disease outcomes. Most importantly, because of this property, this model will be one of the first in which we can directly test, at the level of a whole organism, if ablation of expression of the toxic RNA after a period of exposure can reverse its toxic effects. All DM patients have endured exposure to the toxic RNA from birth and thus a model such as this one will be able to provide valuable and relevant insight into this therapeutic strategy
Keywords: disease /disorder model, gene mutation, gene therapy, genetically modified animal, laboratory mouse, messenger RNA, myotonic dystrophy, small interfering RNA, therapy design /development RNA splicing, cell differentiation, chloride channel, gene expression, myoblast, nonhuman therapy evaluation, nucleic acid repetitive sequence, protein kinase, tetracycline, troponin Lentivirus, biotechnology, fluorescent in situ hybridization, immunocytochemistry, northern blotting, tissue /cell culture, transfection /expression vector, western blotting
Project start date: 2005-09-01
Project end date: 2010-08-31
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Inducible Transgenic Mouse Model Of RNA Toxicity
Mani S Mahadevan, Assistant Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01AR052771-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SMEP
Abstract: Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults. There are two types, DM1 and DM2, both being autosomal dominant disorders caused by expansions of microsatellite repeats within non-coding regions of their respective genes. DM1 is far more common; however both forms of DM are likely to share similar pathogenic mechanisms. The DM1 mutation is an expansion of a CTG triplet repeat in the 3 untranslated region (3 UTR) of the DM protein kinase (DMPK) gene. A prevailing hypothesis in the field is that many aspects of DM are caused by the expression of the mutant mRNA. DM1 and DM2 represent the first examples of toxic RNA mediated disease pathogenesis. We have already developed and characterized extensively, a myoblast cell culture model to clearly demonstrate the toxic effects of the mutant DMPK mRNA on muscle differentiation. To study the hypothesis further, the aims of this proposal are to develop and characterize an inducible transgenic mouse model of RNA toxicity for DM type 1 (DM1) and to develop a siRNA (small interfering RNA) therapeutic approach to get rid of the toxic RNA which can be tested in both our cell culture and transgenic animal models. The development of transgenic mouse models will aid in understanding disease pathogenesis and will also provide a system with which to test out potential therapeutic .strategies. The ability to control gene expression through an inducible system will enable better characterization of and better correlation with the onset and levels of expression of the toxic RNA in DM1 and disease outcomes. Most importantly, because of this property, this model will be one of the first in which we can directly test, at the level of a whole organism, if ablation of expression of the toxic RNA after a period of exposure can reverse its toxic effects. All DM patients have endured exposure to the toxic RNA from birth and thus a model such as this one will be able to provide valuable and relevant insight into this therapeutic strategy.
Keywords: disease /disorder model, gene mutation, gene therapy, genetically modified animal, laboratory mouse, messenger RNA, myotonic dystrophy, small interfering RNA, therapy design /development, RNA splicing, cell differentiation, chloride channel, gene expression, myoblast, nonhuman therapy evaluation, nucleic acid repetitive sequence, protein kinase, tetracycline, troponin, Lentivirus, biotechnology, fluorescent in situ hybridization, immunocytochemistry, northern blotting, tissue /cell culture, transfection /expression vector, western blotting
Project start date: 2005-09-01
Project end date: 2010-08-31
5R01AR052771-03 (2007): $316029
5R01AR052771-02 (2006): $325467
Grants awarded to Mani S Mahadevan
Inducible Transgenic Mouse Model Of RNA Toxicity
Mani S Mahadevan, Assistant Professor
Pathologyuniversity Of Virginia Charlottesville
Grant 5R01AR052771-04 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: SMEP
Abstract: Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults. There are two types, DM1 and DM2, both being autosomal dominant disorders caused by expansions of microsatellite repeats within non-coding regions of their respective genes. DM1 is far more common; however both forms of DM are likely to share similar pathogenic mechanisms. The DM1 mutation is an expansion of a CTG triplet repeat in the 3´ untranslated region (3´UTR) of the DM protein kinase (DMPK) gene. A prevailing hypothesis in the field is that many aspects of DM are caused by the expression of the mutant mRNA. DM1 and DM2 represent the first examples of toxic RNA mediated disease pathogenesis. We have already developed and characterized extensively, a myoblast cell culture model to clearly demonstrate the toxic effects of the mutant DMPK mRNA on muscle differentiation. To study the hypothesis further, the aims of this proposal are to develop and characterize an inducible transgenic mouse model of RNA toxicity for DM type 1 (DM1) and to develop a siRNA (small interfering RNA) therapeutic approach to get rid of the toxic RNA which can be tested in both our cell culture and transgenic animal models. The development of transgenic mouse models will aid in understanding disease pathogenesis and will also provide a system with which to test out potential therapeutic .strategies. The ability to control gene expression through an inducible system will enable better characterization of and better correlation with the onset and levels of expression of the toxic RNA in DM1 and disease outcomes. Most importantly, because of this property, this model will be one of the first in which we can directly test, at the level of a whole organism, if ablation of expression of the toxic RNA after a period of exposure can reverse its toxic effects. All DM patients have endured exposure to the toxic RNA from birth and thus a model such as this one will be able to provide valuable and relevant insight into this therapeutic strategy
Keywords: disease /disorder model, gene mutation, gene therapy, genetically modified animal, laboratory mouse, messenger RNA, myotonic dystrophy, small interfering RNA, therapy design /development RNA splicing, cell differentiation, chloride channel, gene expression, myoblast, nonhuman therapy evaluation, nucleic acid repetitive sequence, protein kinase, tetracycline, troponin Lentivirus, biotechnology, fluorescent in situ hybridization, immunocytochemistry, northern blotting, tissue /cell culture, transfection /expression vector, western blotting
Project start date: 2005-09-01
Project end date: 2010-08-31
1R01AR052771-01 (2005): $335134
MOLECULAR MECHANISMS OF THE MYOTONIC DYSTROPHY MUTATION
Mani S Mahadevan, Assistant Professor
Medical Geneticsuniversity Of Wisconsin Madison
21 N. Park Street, Suite 6401
madison, Wi 537151218
Grant 5R01AR045992-03 from National Institute Of Arthritis And Musculoskeletal And Skin Diseases, IRG: HED
Abstract: Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults with a global incidence of 1 per 8000. The DM mutation was found to be an expansion of an unstable CTG triplet repeat in the 3´ untranslated region (3´UTR) of a gene encoding a serine-threonine protein kinase (DMPK). However, the mechanism by which it causes disease is unknown. We and others have found that the mutant DMPK mRNA is trapped within the nucleus of DM cells and forms distinct, stable foci of mRNA. In addition, we have demonstrated that the mutant DMPK 3´UTR mRNAhas significant negative effects on gene expression. Furthermore, we have identified that expression of the mutant DMPK 3´UTR mRNA in myoblasts causes defects in myoblast fusion and differentiation, demonstrating that this RNA work in trans on the expression of other transcripts, and causes a disease relevant cellular phenotype. This study is directed at understanding the molecular biology of DM by addressing the hypothesis that DM is a paradigm for RNA mediated disease processes. The proposed experiments will be aimed at assessing and determining the effect of the DMPK messenger RNA (mRNA) on gene expression. The effects of the normal and mutant DMPK 3´UTR mRNA will be studied initially at the cellular level, secondly from a biochemical persepective and finally in vivo through the creation of a transgenic murine model. The hypothesis to be tested by this proposal is that Myotonic dystrophy is a disease in which dysregulation of RNA metabolism mediated by the mutant DMPK mRNA contributes to the pathophysiology of DM. The specific aims of this proposal are directed at 1) studying the effects of the DM mutation in a cell culture model, 2) identifying genes whose expression is altered by the presence of the mutant DMPK 3´UTR mRNA and 3) the establishment of a murine model to study the in vivo effects of the DM mutation on RNA metabolism and their contribution to DM pathogenesis. The long term objectives of this proposal are to understand the molecular mechanisms by which the DM mutation functions in order to provide insight into the pathophysiology of DM, to allow for the development of appropriate animal models, and to eventually lead to a more rational approach to therapeutic intervention in DM
Keywords: gene expression, gene mutation, messenger RNA, myotonic dystrophy, nucleic acid metabolism, protein kinase genetic model, model design /development, myoblast, phenotype beta galactosidase, chloramphenicol acetyltransferase, fluorescent in situ hybridization, gene targeting, immunocytochemistry, laboratory mouse, northern blotting, reporter gene, subtraction hybridization, transgenic animal, western blotting
Project start date: 1999-04-09
Project end date: 2004-03-31
5R01AR045992-03 (2001): $273095
5R01AR045992-02 (2000): $265140
1R01AR045992-01 (1999): $257419
Related Publications
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