Design and Analysis of Effects of Triplet Repeat Oligonucleotides in Cell Models for Myotonic Dystrophy

González-Barriga, Anchel; Mulders, Susan A. M.; Giessen, Jeroen van de; Hooijer, Jeroen D; Bijl, Suzanne; Kessel, Ingeborg Dg van; Beers, Josee van; Deutekom, Judith C. van; Fransen, J.A.M.; Wieringa, Bé; Wansink, Derick G.

doi:10.1038/mtna.2013.9

Cited by 40 publications

(47 citation statements)

References 48 publications

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“…Typically, gapmers complementary to the repeat are transfected into cells and induce RNase H cleavage 3, 61-63 . Oligonucleotides that target r(CUG) exp improve DM1-associated defects in a mouse model; however, electrostimulation was required 3, 5 and one caused muscle damage 3 .…”

Section: Resultsmentioning

confidence: 99%

Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

et al. 2013

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Myotonic dystrophy type 2 (DM2) is an untreatable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)exp) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5’CCUG/3’GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA-small molecule interactions. We also report the first crystal structure of r(CCUG)exp refined to 2.35 Å. Structural analysis of the three 5’CCUG/3’GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na+ and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5’CCUG/3’GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops.

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Section: Resultsmentioning

confidence: 99%

Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

et al. 2013

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“…From DM1 cell and mouse model studies, there is significant support for considering the RNA gain-of-function toxicity the prime therapeutic target, and proof-of-concept testing has already demonstrated that antisense oligonucleotide (AON)-mediated degradation of DMPK (CUG)n transcripts or disruption of abnormal RNP complexes by RNA binding or MBNL displacement has potential therapeutic utility 29, 30, 31, 32. Hurdles that still have to be overcome for use in vivo relate to modes of administration, cell-type specificity of action, and possible immune effects of repeated treatment with AONs or small molecule drugs.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

et al. 2017

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Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

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“…21). 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 .…”

Section: Introductionmentioning

confidence: 99%

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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Design and Analysis of Effects of Triplet Repeat Oligonucleotides in Cell Models for Myotonic Dystrophy

Cited by 40 publications

References 48 publications

Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

Structure of the Myotonic Dystrophy Type 2 RNA and Designed Small Molecules That Reduce Toxicity

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

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

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