Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs

François, Virginie; Klein, Arnaud; Beley, Cyriaque; Jollet, Arnaud; Lemercier, Camille; Garcı́a, Luis; Furling, Denis

doi:10.1038/nsmb.1958

Cited by 67 publications

(57 citation statements)

References 13 publications

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“…We sought to determine if the treatment that abolished CUGexp foci formation and sequestration of MBNL1 affects the alternative splicing of MBNL1-regulated pre-mRNAs that have been reported to be aberrantly spliced in human DM1 cells 9 , 27 , 48 . For this, we tested the splicing pattern of endogenous SERCA1 exon 22 ( SERCA1 +E22), LDB3 exon 7 ( LDB3 +E7), MBNL1 exon 7 ( MBNL1 +E7), and DMD exon 78 ( DMD +E78) by RT-PCR in proliferating human myoblasts.…”

Section: Resultsmentioning

confidence: 99%

“…The antisense technology that utilizes morpholino CAG-25 oligonucleotides, 22 , 23 other chemically modified CAG repeat antisense oligonucleotides, 24 , 25 and synthetic siRNAs to target CUG repeats 26 appears to be effective in DM1 cells and mouse models of the disease. Additionally, viral vector-mediated expression of hU7-snRNA-(CAG) has shown to be beneficial in DM1 myoblasts 27 . Also, several bioactive small molecules that are CUG repeat binders have been reported as potential therapeutic agents for DM1 and are able to inhibit the interactions between expanded CUG RNA and MBNL1 protein 28 – 33 .…”

Section: Introductionmentioning

confidence: 99%

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Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

et al. 2014

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Expandable (CTG)n repeats in the 3′ UTR of the DMPK gene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the size and number of foci containing expanded CUG repeat transcripts, decreased steady-state levels of CUGBP1 protein, and consequent improvement of the aberrant alternative splicing of several pre-mRNAs misregulated in DM1.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

et al. 2014

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“…8–13 As these treatments advance to clinical trials, there is a pressing need for biomarkers to assess disease progression and therapeutic response. Splicing defects served this purpose during the preclinical phases of drug development.…”

Section: Introductionmentioning

confidence: 99%

Splicing biomarkers of disease severity in myotonic dystrophy

Nakamori

Sobczak

Puwanant

et al. 2013

Annals of Neurology

209

299

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Objective To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2). Methods In a discovery cohort we used microarrays to perform global analysis of alternative splicing in DM1 and DM2. The newly identified splicing changes were combined with previous data to create a panel of 50 putative splicing defects. In a validation cohort of 50 DM1 subjects we measured the strength of ankle dorsiflexion (ADF) and then obtained a needle biopsy of tibialis anterior (TA) to analyze splice events in muscle RNA. The specificity of DM-associated splicing defects was assessed in disease controls. The CTG expansion size in muscle tissue was determined by Southern blot. The reversibility of splicing defects was assessed in transgenic mice by using antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. Results Forty-two splicing defects were confirmed in TA muscle in the validation cohort. Among these, 20 events showed graded changes that correlated with ADF weakness. Five other splice events were strongly affected in DM1 subjects with normal ADF strength. Comparison to disease controls and mouse models indicated that splicing changes were DM-specific, mainly attributable to MBNL1 sequestration, and reversible in mice by targeted knockdown of toxic RNA. Splicing defects and weakness were not correlated with CTG expansion size in muscle tissue. Interpretation Alternative splicing changes in skeletal muscle may serve as biomarkers of disease severity and therapeutic response in myotonic dystrophy.

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“…Various approaches have been taken to silence toxic RNA or protein in microsatellite expansion diseases, including antisense oligonucleotides (Wheeler et al, 2012, Lagier-Tourenne et al, 2013, Kanadia et al, 2003, Sah & Aronin, 2011), small RNAs (Furling et al, 2003, François et al, 2011, Langlois et al, 2003, Harper et al, 2005), and small molecules (Siboni et al, 2015, Rzuczek et al, 2017). Perturbation to co-factors of RNA polymerase II (RNA Pol II) reduces transcription through expanded repeats in HD (Liu et al, 2012) and C9ALS/FTD (Kramer et al, 2016) models, and treatment with Actinomycin D at nanomolar doses preferentially impedes transcription of CTG repeats in DM models (Siboni et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

et al. 2017

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Summary Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs' endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

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Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs

Cited by 67 publications

References 13 publications

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Splicing biomarkers of disease severity in myotonic dystrophy

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

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