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Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy.

ABSTRACT: Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast-myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2'-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

SUBMITTER: Mulders SA

PROVIDER: S-EPMC2728995 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Triplet repeat-derived siRNAs enhance RNA-mediated toxicity in a Drosophila model for myotonic dystrophy.

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Project description:Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells.

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