DM Cells Research Articles

Small Molecule Screening Identifies HSP90 as a Modifier of RNA Foci in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the DMPK gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control DMPK expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of DMPK mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of DMPK mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of DMPK mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of DMPK mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for DMPK mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

Peptide-conjugated antimiRs improve myotonic dystrophy type 1 phenotypes by promoting endogenous MBNL1 expression

Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the DMPK gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In HSALR mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.

BlockmiR AONs as Site-Specific Therapeutic MBNL Modulation in Myotonic Dystrophy 2D and 3D Muscle Cells and HSALR Mice.

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 DM1 protein kinase (DMPK) gene that, upon transcription, physically sequesters the Muscleblind-like (MBNL) family of splicing regulator proteins. The high-affinity binding occurring between the proteins and the repetitions disallow 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 on previously demonstrated evidence showing that the silencing of miRNA-23b and miRNA-218 can increase MBNL1 protein in DM1 cells and mice. Here, we use blockmiR antisense technology in DM1 muscle cells, 3D mouse-derived muscle tissue, and in vivo mice to block the binding sites of these microRNAs in order to increase MBNL translation into protein without binding to microRNAs. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and highly specific transcriptomic expression. The blockmiRs are well tolerated in 3D mouse skeletal tissue inducing no immune response. In vivo, a candidate blockmiR also increases Mbnl1/2 protein and rescues grip strength, splicing, and histological phenotypes.

Establishment of quantitative and consistent in vitro skeletal muscle pathological models of myotonic dystrophy type 1 using patient-derived iPSCs

Myotonic dystrophy type 1 (DM1) is caused by expanded CTG repeats (CTGexp) in the dystrophia myotonica protein kinase (DMPK) gene, and the transcription products, expanded CUG repeats, sequester muscleblind like splicing regulator 1 (MBNL1), resulting in the nuclear MBNL1 aggregation in the DM1 cells. Loss of MBNL1 function is the pivotal mechanism underlying the pathogenesis of DM1. To develop therapeutics for DM1, proper human in vitro models based on the pathologic mechanism of DM1 are required. In this study, we established robust in vitro skeletal muscle cell models of DM1 with patient-derived induced pluripotent stem cells (iPSCs) using the MyoD1-induced system and iPSCs-derived muscle stem cell (iMuSC) differentiation system. Our newly established DM1 models enable simple quantitative evaluation of nuclear MBNL1 aggregation and the downstream splicing defects. Quantitative analyses using the MyoD1-induced myotubes showed that CTGexp-deleted DM1 skeletal myotubes exhibited a reversal of MBNL1-related pathologies, and antisense oligonucleotide treatment recovered these disease phenotypes in the DM1-iPSCs-derived myotubes. Furthermore, iMuSC-derived myotubes exhibited higher maturity than the MyoD1-induced myotubes, which enabled us to recapitulate the SERCA1 splicing defect in the DM1-iMuSC-derived myotubes. Our quantitative and reproducible in vitro models for DM1 established using human iPSCs are promising for drug discovery against DM1.

Sustainable recovery of MBNL activity in autoregulatory feedback loop in myotonic dystrophy

Muscleblind-like proteins (MBNLs) are RNA-binding proteins essential for the developmental regulation of various processes including alternative splicing. Their activity is misregulated in myotonic dystrophy type 1 (DM1), an incurable genetic, neuro-muscular disorder caused by uncontrolled expansion of CTG repeats. Mutant RNAs containing hundreds or thousands of repeats efficiently sequester MBNL proteins. As a consequence, global alternative splicing abnormalities are induced. Importantly, the size of expansion differs significantly not only between patients but also between different parts of the same muscle as a consequence of somatic expansion. One of the potential therapeutic strategies in DM is overexpression of MBNLs. However, gene therapy tools might induce excessive activity of MBNLs, what in turn might change the metabolism of many RNAs. To overcome these limitations, we designed an autoregulated MBNL1 overexpression system. The genetic construct contains an MBNL1-coding sequence separated by the fragment of ATP2A1 pre-mRNA with an MBNL-sensitive alternative exon containing stop codon in the reading frame of MBNL1. Inclusion of this exon leads to the arrangement of an inactive form of the protein, but exclusion gives rise to fully active MBNL1. This approach enables the autoregulation of the amount of overexpressed MBNL1 with high dynamic range which ensures a homogeneous level of this protein in cells treated with the genetic construct. We demonstrated beneficial effects of an autoregulated construct on alternative splicing patterns in DM1 models and cells derived from patients with DM1.

Delay of EGF-Stimulated EGFR Degradation in Myotonic Dystrophy Type 1 (DM1).

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the 3′ untranslated region of the dystrophia myotonica protein kinase gene. AKT dephosphorylation and autophagy are associated with DM1. Autophagy has been widely studied in DM1, although the endocytic pathway has not. AKT has a critical role in endocytosis, and its phosphorylation is mediated by the activation of tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR). EGF-activated EGFR triggers the internalization and degradation of ligand–receptor complexes that serve as a PI3K/AKT signaling platform. Here, we used primary fibroblasts from healthy subjects and DM1 patients. DM1-derived fibroblasts showed increased autophagy flux, with enlarged endosomes and lysosomes. Thereafter, cells were stimulated with a high concentration of EGF to promote EGFR internalization and degradation. Interestingly, EGF binding to EGFR was reduced in DM1 cells and EGFR internalization was also slowed during the early steps of endocytosis. However, EGF-activated EGFR enhanced AKT and ERK1/2 phosphorylation levels in the DM1-derived fibroblasts. Therefore, there was a delay in EGF-stimulated EGFR endocytosis in DM1 cells; this alteration might be due to the decrease in the binding of EGF to EGFR, and not to a decrease in AKT phosphorylation.

Development of Therapeutic Approaches for Myotonic Dystrophies Type 1 and Type 2.

Myotonic Dystrophies type 1 (DM1) and type 2 (DM2) are complex multisystem diseases without disease-based therapies. These disorders are caused by the expansions of unstable CTG (DM1) and CCTG (DM2) repeats outside of the coding regions of the disease genes: DMPK in DM1 and CNBP in DM2. Multiple clinical and molecular studies provided a consensus for DM1 pathogenesis, showing that the molecular pathophysiology of DM1 is associated with the toxicity of RNA CUG repeats, which cause multiple disturbances in RNA metabolism in patients’ cells. As a result, splicing, translation, RNA stability and transcription of multiple genes are misregulated in DM1 cells. While mutant CCUG repeats are the main cause of DM2, additional factors might play a role in DM2 pathogenesis. This review describes current progress in the translation of mechanistic knowledge in DM1 and DM2 to clinical trials, with a focus on the development of disease-specific therapies for patients with adult forms of DM1 and congenital DM1 (CDM1).

Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing

ABSTRACTSkeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1.

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy.

Transcriptome Analysis Reveals Altered Inflammatory Pathway in an Inducible Glial Cell Model of Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1), the most frequent inherited muscular dystrophy in adults, is caused by the CTG repeat expansion in the 3′UTR of the DMPK gene. Mutant DMPK RNA accumulates in nuclear foci altering diverse cellular functions including alternative splicing regulation. DM1 is a multisystemic condition, with debilitating central nervous system alterations. Although a defective neuroglia communication has been described as a contributor of the brain pathology in DM1, the specific cellular and molecular events potentially affected in glia cells have not been totally recognized. Thus, to study the effects of DM1 mutation on glial physiology, in this work, we have established an inducible DM1 model derived from the MIO-M1 cell line expressing 648 CUG repeats. This new model recreated the molecular hallmarks of DM1 elicited by a toxic RNA gain-of-function mechanism: accumulation of RNA foci colocalized with MBNL proteins and dysregulation of alternative splicing. By applying a microarray whole-transcriptome approach, we identified several gene changes associated with DM1 mutation in MIO-M1 cells, including the immune mediators CXCL10, CCL5, CXCL8, TNFAIP3, and TNFRSF9, as well as the microRNAs miR-222, miR-448, among others, as potential regulators. A gene ontology enrichment analyses revealed that inflammation and immune response emerged as major cellular deregulated processes in the MIO-M1 DM1 cells. Our findings indicate the involvement of an altered immune response in glia cells, opening new windows for the study of glia as potential contributor of the CNS symptoms in DM1.

Single-cell RNA Sequencing Deciphers Immune Landscape of Human Recurrent Miscarriage

Single-cell RNA Sequencing Deciphers Immune Landscape of Human Recurrent Miscarriage

The Dimeric Form of 1,3-Diaminoisoquinoline Derivative Rescued the Mis-splicing of Atp2a1 and Clcn1 Genes in Myotonic Dystrophy Type 1 Mouse Model.

Expanded CUG repeat RNA in the dystrophia myotonia protein kinase (DMPK) gene causes myotonic dystrophy type 1 (DM1) and sequesters RNA processing proteins, such as the splicing factor muscleblind‐like 1 protein (MBNL1). Sequestration of splicing factors results in the mis‐splicing of some pre‐mRNAs. Small molecules that rescue the mis‐splicing in the DM1 cells have drawn attention as potential drugs to treat DM1. Herein we report a new molecule JM642 consisted of two 1,3‐diaminoisoquinoline chromophores having an auxiliary aromatic unit at the C5 position. JM642 alternates the splicing pattern of the pre‐mRNA of the Ldb3 gene in the DM1 cell model and Clcn1 and Atp2a1 genes in the DM1 mouse model. In vitro binding analysis by surface plasmon resonance (SPR) assay to the r(CUG) repeat and disruption of ribonuclear foci in the DM1 cell model suggested the binding of JM642 to the expanded r(CUG) repeat in vivo, eventually rescue the mis‐splicing.

Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion.

Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington's disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD-derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders.

CDK12 inhibition reduces abnormalities in cells from patients with myotonic dystrophy and in a mouse model.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

FISH Protocol for Myotonic Dystrophy Type 1 Cells.

Mutant DMPK transcripts containing expanded CUG repeats (CUGexp) are retained within the nucleus of myotonic dystrophy type 1 (DM1) cells as discrete foci. Nuclear CUGexp-RNA foci that sequester MBNL1 splicing factor represent a hallmark of this RNA dominant disease caused by the expression of expanded microsatellite repeats. Here we described fluorescent in situ hybridization (FISH) techniques to detect either RNA containing CUG expansion or DMPK transcripts in human DM1 or WT cells. In addition, we propose a combined FISH/immunofluorescence protocol to visualize the colocalization of MBNL1 with CUGexp-RNA foci in DM1 cells.

Intrinsically cell-penetrating multivalent and multitargeting ligands for myotonic dystrophy type 1

Developing highly active, multivalent ligands as therapeutic agents is challenging because of delivery issues, limited cell permeability, and toxicity. Here, we report intrinsically cell-penetrating multivalent ligands that target the trinucleotide repeat DNA and RNA in myotonic dystrophy type 1 (DM1), interrupting the disease progression in two ways. The oligomeric ligands are designed based on the repetitive structure of the target with recognition moieties alternating with bisamidinium groove binders to provide an amphiphilic and polycationic structure, mimicking cell-penetrating peptides. Multiple biological studies suggested the success of our multivalency strategy. The designed oligomers maintained cell permeability and exhibited no apparent toxicity both in cells and in mice at working concentrations. Furthermore, the oligomers showed important activities in DM1 cells and in a DM1 liver mouse model, reducing or eliminating prominent DM1 features. Phenotypic recovery of the climbing defect in adult DM1 Drosophila was also observed. This design strategy should be applicable to other repeat expansion diseases and more generally to DNA/RNA-targeted therapeutics.

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes.

Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. DM is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of DM in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2, which is characterized by nucleotide repeat expansions often greater than 5,000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, in the presence of muscleblind-like 1 (MBNL1) foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls and DM1 and DM2 subjects, and we differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. iPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High-resolution imaging revealed tight association between MBNL clusters and RNA foci in DM1. Ca2+ transients differed between DM1- and DM2 iPSC-derived cardiomyocytes, and each differed from healthy control cells. RNA-sequencing from DM1- and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression, as well as differential aberrant splicing patterns. Together, these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.

Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are human neuromuscular disorders associated with mutations of simple repetitive sequences in affected genes. The abnormal expansion of CTG repeats in the 3′-UTR of the DMPK gene elicits DM1, whereas elongated CCTG repeats in intron 1 of ZNF9/CNBP triggers DM2. Pathogenesis of both disorders is manifested by nuclear retention of expanded repeat-containing RNAs and aberrant alternative splicing. The precise determination of absolute numbers of mutant RNA molecules is important for a better understanding of disease complexity and for accurate evaluation of the efficacy of therapeutic drugs. We present two quantitative methods, Multiplex Ligation-Dependent Probe Amplification and droplet digital PCR, for studying the mutant DMPK transcript (DMPKexpRNA) and the aberrant alternative splicing in DM1 and DM2 human tissues and cells. We demonstrate that in DM1, the DMPKexpRNA is detected in higher copy number than its normal counterpart. Moreover, the absolute number of the mutant transcript indicates its low abundance with only a few copies per cell in DM1 fibroblasts. Most importantly, in conjunction with fluorescence in-situ hybridization experiments, our results suggest that in DM1 fibroblasts, the vast majority of nuclear RNA foci consist of a few molecules of DMPKexpRNA.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

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.