CRISPRi; MT: RNA/DNA Editing; gene silencing; gene therapy; myotonic dystrophy type 1; neuromuscular disease; Molecular Medicine; Drug Discovery
Abstract :
[en] Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.
Disciplines :
Neurology
Author, co-author :
Porquet, Florent ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Pharmacologie ; Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France
Weidong, Lin; Laboratory of Molecular and Cellular Epigenetics, GIGA-Cancer, ULiège, 4000 Liège, Belgium
Jehasse, Kevin ; Université de Liège - ULiège > GIGA > GIGA Neurosciences - Neurophysiology
Gazon, Hélène ; Université de Liège - ULiège > GIGA > GIGA Cancer - Cellular and Molecular Epigenetics
Kondili, Maria; Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France
Blacher, Silvia ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Biologie cellulaire et moléculaire
Massotte, Laurent ; Université de Liège - ULiège > Département des sciences biomédicales et précliniques
Furling, Denis; Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France
Gillet, Nicolas ; Université de Liège - ULiège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Microbial technologies ; Namur Research Institute for Life Sciences (NARILIS), Integrated Veterinary Research Unit (URVI), University of Namur, 5000 Namur, Belgium
Klein, Arnaud François ✱; Sorbonne Université, INSERM, Institut de Myologie, Centre de Recherche en Myologie, 75013 Paris, France
Seutin, Vincent ✱; Université de Liège - ULiège > Département des sciences biomédicales et précliniques > Pharmacologie
Willems, Luc ✱; Université de Liège - ULiège > GIGA > GIGA Cancer - Cellular and Molecular Epigenetics
The authors thank the human cell immortalization facility of the Myology Institute (Paris, France) as well as the GIGA technological platforms for support. In particular, the authors are grateful to François Giroulle, Alexandre Hego, Latifa Karim, Sandra Ormenese, Wouter Coppieters, Alexandra Revnic, Arnaud Lavergne, Raafat Stephan, and Stephen Freeman for their technical expertise and engagement, as well as Mario Gomes-Pereira, Marc Bitoun, and Geneviève Gourdon for their advice. This work was supported by the Fonds National de la Recherche Scientifique (FNRS, FRIA grant), The Myotonic Dystrophy Foundation and Wyck Foundations (2018 PhD trainee fellowship in Myotonic Dystrophy), the Association Belge contre les Maladies Neuro-Musculaires (ABMM; call 2019–2020), and the Fondation Léon Fredericq (FLF). F.P. was supported by fellowships of the Fonds National de la Recherche Scientifique , The Myotonic Dystrophy Foundation , and Wyck Foundations . V.S. is a full professor at ULiege, while L. Willems is a research director at the FNRS and A.F.K. is a research scientist at INSERM.The authors thank the human cell immortalization facility of the Myology Institute (Paris, France) as well as the GIGA technological platforms for support. In particular, the authors are grateful to François Giroulle, Alexandre Hego, Latifa Karim, Sandra Ormenese, Wouter Coppieters, Alexandra Revnic, Arnaud Lavergne, Raafat Stephan, and Stephen Freeman for their technical expertise and engagement, as well as Mario Gomes-Pereira, Marc Bitoun, and Geneviève Gourdon for their advice. This work was supported by the Fonds National de la Recherche Scientifique (FNRS, FRIA grant), The Myotonic Dystrophy Foundation and Wyck Foundations (2018 PhD trainee fellowship in Myotonic Dystrophy), the Association Belge contre les Maladies Neuro-Musculaires (ABMM; call 2019–2020), and the Fondation Léon Fredericq (FLF). F.P. was supported by fellowships of the Fonds National de la Recherche Scientifique, The Myotonic Dystrophy Foundation, and Wyck Foundations. V.S. is a full professor at ULiege, while L. Willems is a research director at the FNRS and A.F.K. is a research scientist at INSERM. Conceptualization, F.P.; methodology, F.P.; software, S.B. and M.K.; formal analysis, F.P. and K.J.; investigation, F.P. L. Weidong, A.F.K. H.G. and L.M.; resources, E.D.V. D.F. and A.F.K.; writing – original draft, F.P.; writing – review & editing, F.P. A.F.K. D.F. V.S. and L. Willems; visualization, F.P. and A.F.K.; supervision, A.F.K. N.A.G. L. Willems, and V.S.; project administration, V.S. and L. Willems; funding acquisition, F.P. The authors declare no competing interests.
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