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Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 3' untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats ( CUGexp ). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK -promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp -RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physiological parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK -promoter silencing by CRISPRi as a promising therapeutic approach for DM1., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

CTG repeat expansion (CTG exp ) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTG exp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTG exp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTG exp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). 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 morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient-derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

Myotonic dystrophy type 1 (DM1 also known as Steinert disease) is a multisystemic disorder mainly characterized by myotonia, progressive muscle weakness and wasting, cognitive impairments, and cardiac defects. This autosomal dominant disease is caused by the expression of nuclear retained RNAs containing pathologic expanded CUG repeats that alter the function of RNA-binding proteins in a tissue-specific manner, leading ultimately to neuromuscular dysfunction and clinical symptoms. Although considerable knowledge has been gathered on myotonic dystrophy since its first description, the development of novel relevant disease models remains of high importance to investigate pathophysiologic mechanisms and to assess new therapeutic approaches. In addition to animal models, in vitro cell cultures provide a unique resource for both fundamental and translational research. This review discusses how cellular models broke ground to decipher molecular basis of DM1 and describes currently available cell models, ranging from exogenous expression of the CTG tracts to variable patients' derived cells.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are autosomal dominant neuromuscular diseases caused by microsatellite expansions and belong to the family of RNA-dominant disorders. Availability of cellular models in which the DM mutation is expressed within its natural context is essential to facilitate efforts to identify new therapeutic compounds. Here, we generated immortalized DM1 and DM2 human muscle cell lines that display nuclear RNA aggregates of expanded repeats, a hallmark of myotonic dystrophy. Selected clones of DM1 and DM2 immortalized myoblasts behave as parental primary myoblasts with a reduced fusion capacity of immortalized DM1 myoblasts when compared with control and DM2 cells. Alternative splicing defects were observed in differentiated DM1 muscle cell lines, but not in DM2 lines. Splicing alterations did not result from differentiation delay because similar changes were found in immortalized DM1 transdifferentiated fibroblasts in which myogenic differentiation has been forced by overexpression of MYOD1. As a proof-of-concept, we show that antisense approaches alleviate disease-associated defects, and an RNA-seq analysis confirmed that the vast majority of mis-spliced events in immortalized DM1 muscle cells were affected by antisense treatment, with half of them significantly rescued in treated DM1 cells. Immortalized DM1 muscle cell lines displaying characteristic disease-associated molecular features such as nuclear RNA aggregates and splicing defects can be used as robust readouts for the screening of therapeutic compounds. Therefore, immortalized DM1 and DM2 muscle cell lines represent new models and tools to investigate molecular pathophysiological mechanisms and evaluate the in vitro effects of compounds on RNA toxicity associated with myotonic dystrophy mutations., (© 2017. Published by The Company of Biologists Ltd.)

For the post laser treatment of couperosis a new dermal formulation was developed combining three actives: vitamin K1, A1 and rutin, where both vitamins were incorporated into solid lipid nanoparticles (SLN) and the poorly soluble antioxidant rutin formulated as nanocrystal. All three formulations were stable over 6 months either on their own or after their incorporation into a hydrogel. Vitamin A1 at 0.3% in emulsions shows local skin irritation due to very rapid release. By forming SLN, prolonged release with less irritation potential but deeper penetration was achieved in porcine ear skin. Due to the nanosized rutin, the new hydrogel showed clearly increased antioxidant activity, representing a stronger protection potential against reactive oxygen species (ROS), compared to marketed anti-redness products with rutin as raw drug powder or water-soluble derivative. In addition, rutin nanocrystals showed up to 5 times pronounced penetration compared to μm-sized raw drug powder. The orientating in-vivo case study revealed a three to six times faster recovery after laser treatment of couperosis by twice daily application of the new hydrogel, regarding scabbed-over areas and erythema. Continued use of the new gel also showed preventive properties against recurrences of veins for at least 8 month., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Myotonic Dystrophy (DM), one of the most common neuromuscular disorders in adults, comprises two genetically distinct forms triggered by unstable expanded repeats in non-coding regions. The most common DM1 is caused by expanded CTG repeats in the 3'UTR of the DMPK gene, whereas DM2 is due to large expanded CCTG repeats in the first intron of the CNBP gene. Both mutations induce a pathogenic RNA gain-of-function mechanism. Mutant RNAs containing CUG or CCUG expanded repeats, which are retained in the nuclei as aggregates alter activities of alternative splicing regulators such as MBNL proteins and CELF1. As a consequence, alternative splicing misregulations of several pre-mRNAs are associated with DM clinical symptoms. Currently, there is no available cure for this dominant neuromuscular disease. Nevertheless, promising therapeutic strategies have been developed in the last decade. Preclinical progress in DM research prompted the first DM1 clinical trial based on antisense oligonucleotides promoting a RNase-H-mediated degradation of the expanded CUG transcripts. The ongoing Phase 1/2a clinical trial will hopefully give further insights into the quest to find a bona fide cure for DM1. In this review, we will provide an overview of the different strategies that were developed to neutralize the RNA toxicity in DM1. Different approaches including antisense oligonucleotide technologies, gene therapies or small molecules have been tested and validated in cellular and animal models. Remaining challenges and additional avenues to explore will be discussed.

Myotonic Dystrophy type I (DM1) is caused by an abnormal expansion of CTG triplets in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene, leading to the aggregation of the mutant transcript in nuclear RNA foci. The expanded mutant transcript promotes the sequestration of the MBNL1 splicing factor, resulting in the misregulation of a subset of alternative splicing events. In this study, we identify the DEAD-box RNA helicase p68 (DDX5) in complexes assembled onto in vitro-transcribed CUG repeats. We showed that p68 colocalized with RNA foci in cells expressing the 3'UTR of the DMPK gene containing expanded CTG repeats. We found that p68 increased MBNL1 binding onto pathological repeats and the stem-loop structure regulatory element within the cardiac Troponin T (TNNT2) pre-mRNA, splicing of which is misregulated in DM1. Mutations in the helicase core of p68 prevented both the stimulatory effect of the protein on MBNL1 binding and the colocalization of p68 with CUG repeats, suggesting that remodeling of RNA secondary structure by p68 facilitates MBNL1 binding. We also found that the competence of p68 for regulating TNNT2 exon 5 inclusion depended on the integrity of MBNL1 binding sites. We propose that p68 acts as a modifier of MBNL1 activity on splicing targets and pathogenic RNA.

Expansion of repeated sequences in non-coding regions of different genes causes a number of inherited diseases including myotonic dystrophies, Huntington disease-like 2, Fragile X tremor/ataxia syndrome and spinocerebellar ataxia 8, 10, 12, 31. Involvement of an RNA gain-of-function mechanism in pathological case has been described and studied in-depth in myotonic dystrophy type 1 (DM1). This inherited neuromuscular disorder is caused by a (CTG)n >50 expansion in the 3' non-coding region of the dystrophia myotonica-protein kinase (DMPK) gene. Expanded CUG transcripts (CUGexp-RNAs) are sequestered in the nucleus within small aggregates and interfere with the regulatory splicing activities of MBNL1 and CELF1 RNA-binding proteins, leading to the misregulation of the alternative splicing of several transcripts. Despite the relevance of aberrant splicing events in this complex pathology, the CUGexp-RNAs trans-dominant effects alter other splicing-independent processes that may also contribute to DM1 pathogenesis. This review will focus on toxic RNA gain-of-function as a pathologic mechanism for DM1 and other repeat expansion disorders., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.

We describe a function for modified human U7 small nuclear RNAs (hU7-snRNAs) distinct from modification of pre-mRNA splicing events. Engineered hU7-snRNAs containing a poly-CAG antisense sequence targeting the expanded CUG repeats of mutant DMPK transcripts in myotonic dystrophy caused specific degradation of pathogenic DMPK mRNAs without affecting the products of wild-type DMPK alleles. Abolition of the RNA gain-of-function toxicity that is responsible for pathogenesis supports the use of hU7-snRNAs for gene silencing in RNA-dominant disorders in which expanded repeats are expressed.

A CTG repeat amplification is responsible for the dominantly inherited neuromuscular disorder, myotonic dystrophy type 1 (DM1), which is characterized by progressive muscle wasting and weakness. The expanded (CTG)n tract not only alters the myogenic differentiation of the DM1 muscle precursor cells but also reduces their proliferative capacity. In this report, we show that these muscle precursor cells containing large CTG expansion sequences have not exhausted their proliferative capacity, but have entered into premature senescence. We demonstrate that an abnormal accumulation of p16 is responsible for this defect because the abolition of p16 activity overcomes early growth arrest and restores an extended proliferative capacity. Our results suggest that the accelerated telomere shortening measured in DM1 cells does not contribute to the aberrant induction of p16. We propose that a cellular stress related to the amplified CTG repeat promotes premature senescence mediated by a p16-dependent pathway in DM1 muscle precursor cells. This mechanism is responsible for the reduced proliferative capacity of the DM1 muscle precursor cells and could participate in both the impaired regeneration and atrophy observed in the DM1 muscles containing large CTG expansions.

Expansions of a (GCN)10/polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) cause autosomal dominant oculopharyngeal muscular dystrophy (OPMD). In OPMD muscles, as in models, PABPN1 accumulates in intranuclear inclusions (INIs) whereas in other diseases caused by similar polyalanine expansions, the mutated proteins have been shown to abnormally accumulate in the cytoplasm. This study presents the impact on the subcellular localization of PABPN1 produced by large expansions or deletion of its polyalanine tract. Large tracts of more than 24 alanines result in the nuclear accumulation of PABPN1 in SFRS2-positive functional speckles and a significant decline in cell survival. These large expansions do not cause INIs formation nor do they lead to cytoplasmic accumulation. Deletion of the polyalanine tract induces the formation of aggregates that are located on either side and cross the nuclear membrane, highlighting the possible role of the N-terminal polyalanine tract in PABPN1 nucleo-cytoplasmic transport. We also show that even though five other proteins with polyalanine tracts tend to aggregate when over-expressed they do not co-aggregate with PABPN1 INIs. This study presents the first experimental evidence that there may be a relative loss of function in OPMD by decreasing the availability of PABPN1 through an INI-independent mechanism.

Oculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a (GCN)10 to a (GCN)11-17 repeat coding for a polyalanine domain at the N-terminal part of poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by the presence of intranuclear inclusions (INIs) in skeletal muscle fibers of patients. The formation of GFP-b13AlaPABPN1 INIs and their fate through the cell cycle were followed by time-lapse imaging. Our observations demonstrated that the GFP-b13AlaPABPN1 INIs are dynamic structures that can disassemble during mitosis. However, their presence in cells occasionally led to apoptosis. The length of the polyalanine tail or the overexpression of PABPN1 did not significantly affect the percentage of soluble PABPN1 in vitro. Moreover, overexpression of either the wild type (wt) or mutant (mut) forms of PABPN1 slowed down the cell proliferation. The slowing down of proliferation together with the occasional occurrence of apoptosis could contribute in vivo to the late onset of this disease.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.