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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE173359 | GEO | 2022/07/31

REPOSITORIES: GEO

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Project description:For some neurological disorders, disease is primarily RNA-mediated due to expression of non-coding microsatellite expansion RNAs (RNAexp). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNAexp in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases. HITS-CLIP analysis was performed to identify RNA binding sites of MBNL2 in control, DM type 1 (DM1), and DM type 2 (DM2) autopsy-derived brain (n=3). Two regions of the brain selected for the study included the frontal cortex and hippocampus. Libraries were sequenced and wiggle files were generated for each biological replicate as well as three pooled biological replicates (3BRs) per group (control, DM1, and DM2). In addition, differential CLIP analysis (dCLIP) was performed to normalize binding data between groups and identify statistically significant changes in binding. The dCLIP analysis generated bedgraph files representing normalized binding profiles of MBNL2 in each group (control, DM1, and DM2) for visualization and comparative analysis. PolyA-seq was performed on control, DM1, and DM2 autopsy-derived brain samples (hippocampus and frontal cortex, n=3) as well as wild-type (WT) and Mbnl1; Mbnl2 conditional double knockout (Mbnl1-/-; Mbnl2c/c; Nestin-Cre+/- or DKO) brain (n=3). Libraries were sequenced and the resultant files were processed and aligned to the reference genomes (hg19 and mm10). Further computational processing was performed to remove internal oligo(dT) mis-priming events, identify valid polyA sites, and trim to the exact polyA sites. BedGraph files were generated for each group in human (control, DM1, and DM2) and mouse (WT and Mbnl DKO) for comparative visualization.

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

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