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Enhanced antisense oligonucleotide delivery reveals that transcript turnover impacts apparent splicing rescue in myotonic dystrophy

ABSTRACT: Steric-blocking antisense oligonucleotides rescue Myotonic dystrophy type 1 phenotypes in preclinical models and are under evaluation in clinical trials. However, rationale for biomarker selection remains a topic of debate. Here, we show that a cyclic cell-penetrating peptide that escapes endosomes enhances muscle delivery of a phosphorodiamidate morpholino oligonucleotide (PMO) designed to block pathogenic CUG repeat expansions in HSALR mice. A single systemic administration rescued mis-splicing and eliminated myotonia one-week post-injection, with partial splicing rescue evident after 24 hours. Interestingly, some exons showed more robust rescue than others, but relationships between MBNL concentration ([MBNL]) and Percent Spliced In (Ψ) could not fully explain extent of rescue. We hypothesized that since pre-existing transcripts must be degraded to reveal full drug effect, rates of transcript replacement might account for these discrepancies. We formulated a mathematical framework and used Bayesian inference to model how apparent Ψ lags behind nascent Ψ as a function of time; faster rates of replacement result in shorter lags. In vivo 5-ethynyl uridine labeling followed by RNAseq validated these predictions. Overall, we show that transcript turnover influences Ψ during periods of dynamically changing [MBNL] and recommend that this be considered when selecting splicing biomarkers and interpreting responses to therapeutic interventions.

ORGANISM(S): Mus musculus

PROVIDER: GSE319608 | GEO | 2026/05/28

REPOSITORIES: GEO

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Increased Muscleblind levels by chloroquine treatment improves myotonic dystrophy type 1 phenotypes in in vitro and in vivo models

Project description:Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3'UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately originates phenotypes. In this work we demonstrate that treatment with the anti-autophagic drug chloroquine in Drosophila and mouse (HSALR) models, and patient-derived myoblasts, was sufficient to upregulate MBNL1 and 2 proteins. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine rescued locomotion and average crossectional muscle area, and extended median survival of DM1 flies. In HSALR mice the drug recovered muscular strength and histopathology signs, and reduced myotonia grade. Taken together these results offer a novel means to replenish the critically low levels of MBNLs in DM1.

2019-11-22 | GSE128844 | GEO

Splicing changes in gastrocnemius muscle from 3-month-old HSALR mice

Project description:The initial aim of the study was to determine the mechanisms underlying the positive effect of AICAR on the phenotype of HSALR mice, a well-known model for DM1. We have previously shown that treatment with AICAR reduces myotonia and mis-splicing in the HSALR mice. We here aimed to identify splicing and transcriptional changes in gastrocnemius muscle from HSALR mice treated or not with AICAR, as compared to FVB control mice. The data obtained from untreated mice were then used in the study focused on NMJ alterations and CaMKII deregulation in DM1.

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Proof of concept of blockmiR induced MBNL rescue in Myotonic dystrophy muscle cells

Project description:The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DMPK gene that is correctly transcribed and physically sequesters the MBNL family of proteins. The high-affinity binding occurring between the proteins and the repetitions disallows MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build off of previously demonstrated evidence showing that the silencing of miR-23b and miR-218 can increase MBNL1 and 2 protein in DM1 cells. Here we use blockmiR antisense technology to block the binding sites of these miRNAs in order to increase MBNL translation into protein without binding to the microRNAs in DM1 muscle cells. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and transcriptomic expression proving that this novel and highly specific strategy could be a potentially viable therapy in Vivo.

2022-07-31 | GSE173359 | GEO

MBNL overexpression rescues cardiac phenotypes in a myotonic dystrophy type 1 heart mouse model

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Loss of MBNL Leads to Disruption of Developmentally Regulated Alternative Polyadenylation in RNA-Mediated Disease

Project description:Mapping MBNL-regulated genome-wide alternative polyadenylation: We report that depletion of Mbnl proteins in mouse embryo fibroblasts (MEFs), DM mouse model quadriceps muscle, and DM-autopsy muscle tissue leads to mis-regulation of alternative polyadenylation We compared WT, Mbnl1/2KO, Mbnl1/2KO/3siRNA, and Mbnl1/2KO/scrambled siRNA MEFs (n=2 for each group) to evaluate alternative polyadenylation shifts that occur due to progressive loss of Mbnl proteins. We also compared WT (1 day old, and 4 months old, n=2 each) and HSALR mouse model (4 months old, n=2) of myotonic dystrophy for developmental alternative polyadenylation defects in myotonic dystrophy. Finally, we compared control and DM1 autopsy muscle tissues (n=3) for changes in alternative polyadenylation. We performed HITS-CLIP analysis of binding sites of Mbnl1, Mbnl2 and Mbnl3 in MEFs (n=3 each). We also performed HITS-CLIP analysis for major skeletal muscle Mbnl protein, Mbnl1 in FVB WT adult muscle (4 months, n=3). Finally we performed HITS-CLIP analysis for CPSF6 in WT and Mbnl1/2 KO MEFs (n=3 each) Please note that the 'readme_Table.txt' describes the contents of 'Table S*.xlsx' files, and the readme_method.txt include additional details about experiemenal procedures.

2014-09-05 | E-GEOD-60487 | biostudies-arrayexpress

A therapeutic antisense oligonucleotide encompassing 2′-O-methoxyethyl modification triggers unique perturbation of the transcriptome

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Differentially expressed genes in myotubes derived from MBNL knockouts hiPSCs generated by CRISPR/Cas9.

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2021-08-04 | GSE161897 | GEO

Differentially expressed genes in myotubes derived from MBNL knockouts hiPSCs generated by CRISPR/Cas9

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Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice

Project description:Antisense oligonucleotides (ASOs) strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nuclear-retained mutant DMPK transcripts containing CUG expansions (CUGexp). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated PMO dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison to unconjugated PMO and other ASO strategies. Thus, low dose treatment of Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease-transcriptome.

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