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BACKGROUNDMyotonic dystrophy type 1 (DM1) is a multisystemic, CTG repeat expansion disorder characterized by a slow, progressive decline in skeletal muscle function. A biomarker correlating RNA mis-splicing, the core pathogenic disease mechanism, and muscle performance is crucial for assessing response to disease-modifying interventions. We evaluated the Myotonic Dystrophy Splice Index (SI), a composite RNA splicing biomarker incorporating 22 disease-specific events, as a potential biomarker of DM1 muscle weakness.METHODSTotal RNA sequencing of tibialis anterior biopsies from 58 DM1 participants and 33 unaffected/disease controls was used to evaluate RNA splicing events across the disease spectrum. Targeted RNA sequencing was used to derive the SI from biopsies collected at baseline (n = 52) or a 3-month (n = 37) follow-up visit along with clinical measures of muscle performance.RESULTSThe SI demonstrated significant associations with measures of muscle strength and ambulation, including ankle dorsiflexion (ADF) strength and 10-meter run/fast walk (Pearson's r = -0.719 and -0.680, respectively). The SI was relatively stable over 3 months (intraclass correlation coefficient [ICC] = 0.863). Latent-class analysis identified 3 DM1 subgroups stratified by baseline SI (SIMild, SIModerate, and SISevere); SIModerate individuals had a significant increase in the SI over 3 months. Multiple linear regression modeling revealed that baseline ADF and SI were predictive of strength at 3 months (adjusted R² = 0.830).CONCLUSIONThe SI is a reliable biomarker that captures associations of RNA mis-splicing with physical strength and mobility and has prognostic utility to predict future function, establishing it as a potential biomarker for assessment of therapeutic target engagement.TRIAL REGISTRATIONClinicalTrials.gov NCT03981575.FUNDINGFDA (7R01FD006071), Myotonic Dystrophy Foundation, Wyck Foundation, Muscular Dystrophy Association, Novartis, Dyne, Avidity, PepGen, Takeda, Sanofi Genzyme, Pfizer, Arthex, and Vertex Pharmaceuticals.

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 an autosomal dominant inherited multi-system disorder that affects skeletal muscles but also many other organ systems. Molecular targets have been identified and targeted therapies are being developed and tested in first-in-human clinical trials. However, insufficient knowledge of the phenotypic heterogeneity and natural course of the disease, together with a lack of reliable biomarkers, complicate the design of clinical trials. The main objectives of this study are to 1) characterize the phenotypic heterogeneity and disease progression of DM1 in a large cohort; 2) identify baseline characteristics that predict subsequent progression; 3) validate RNA biomarkers of disease severity. This is a prospective, multi-site observational study with a follow-up period of 24 months including approximately 700 adult DM1 patients. Visits will occur at baseline, and months 12 and 24. All patients will undergo strength testing, myotonia assessment, a battery of functional outcome assessments, spirometry, and complete various questionnaires and cognitive tests. Blood and urine samples will be taken at each visit for biomarker studies. A subset of 60 patients will undergo a muscle biopsy at baseline and at an additional 3-month visit. The sensitivity to disease progression and minimally clinically important differences will be determined for the various clinical outcome measures. Associations between baseline patient characteristics and the rate of disease progression will be evaluated. The results of this large international study on DM1 will contribute to optimizing clinical trial design. Both data and biological samples will be collected for future research as well. Clinicaltrials.gov NCT03981575.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disease caused by a CTG repeat expansion in the dystrophia myotonica protein kinase (DMPK) gene. The expanded CUG repeat RNA (CUGexp RNA) transcribed from the mutant allele sequesters the muscleblind-like (MBNL) family of RNA-binding proteins, causing their loss of function and disrupting regulated pre-mRNA processing. We used a DM1 heart mouse model that inducibly expresses CUGexp RNA to test the contribution of MBNL loss to DM1 cardiac abnormalities and explored MBNL restoration as a potential therapy. AAV9-mediated overexpression of MBNL1 and/or MBNL2 significantly rescued DM1 cardiac phenotypes including conduction delays, contractile dysfunction, hypertrophy, and misregulated alternative splicing and gene expression. While robust, the rescue was partial compared with reduced CUGexp RNA and plateaued with increased exogenous MBNL expression. These findings demonstrate that MBNL loss is a major contributor to DM1 cardiac manifestations and suggest that additional mechanisms play a role, highlighting the complex nature of DM1 pathogenesis.

Myotonic dystrophy type 1 (DM1) is a complex, multisystemic neuromuscular disorder with several pathological phenotypes, disease severities and ages of onset. DM1 presents significant challenges in clinical management due to its multisystemic nature, affecting multiple organs and systems beyond skeletal muscle. Tackling this condition requires a comprehensive approach that goes beyond symptom management, particularly considering the complexity of its manifestations and in the delayed diagnosis. In this review we will discuss the multisystem symptoms of DM1 and how this understanding is guiding the development of potential therapies for the improvement of patient outcomes and quality of life. This review aims to explore the available treatments and potential novel disease-modifying therapies targeting DM1 molecular mechanisms to address the broad multisystem symptoms of DM1. Effective strategies to manage symptoms remain crucial, such as physical therapy, medications for myotonia and diligent cardiac care. Metabolic management and hormonal therapies play crucial roles in addressing endocrine and metabolic abnormalities. Nevertheless, promising targeted therapies that include antisense oligonucleotides (ASOs) for RNA degradation, small molecules to disrupt protein-RNA interactions and gene editing offer a prospective approach to the underlying mechanisms of DM1 and improve patient outcomes across the different organ systems.

Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated.

Nuclear-retained transcripts containing expanded repeats are shown to be sensitive to antisense silencing, and in a transgenic mouse model of myotonic dystrophy type 1, systemic administration of ASOs causes a rapid knockdown of the toxic RNA in skeletal muscle, correcting some hallmark features of the disease. A muscular dystrophy rescued by antisense therapy In myotonic dystrophy type I, a common hereditary neuromuscular disorder, the DMPK gene, which normally codes for a protein kinase that is expressed predominantly in skeletal muscle, is mutated in such a way that its transcription results in a toxic RNA molecule that is retained in the nucleus. Charles Thornton and colleagues report the reversal of physical and histological signs of the disease in a mouse model treated with antisense oligonucleotides that caused a rapid knockdown the toxic RNA in skeletal muscle. The effect persisted for up to a year after treatment. Other nuclear-resident RNAs were also sensitive to antisense oligonucleotides, suggesting that this strategy could be used to correct other RNA gain-of-function disorders. Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat 1 , 2 , 3 and are retained in the nucleus 4 , 5 . The mutant RNA exerts a toxic gain-of-function effect 6 , making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target messenger RNAs 7 , 8 . Here we show that nuclear-retained transcripts containing expanded CUG (CUG exp ) repeats are unusually sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUG exp RNA in skeletal muscle, correcting the physiological, histopathologic and transcriptomic features of the disease. The effect was sustained for up to 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1 , a long non-coding RNA (lncRNA) that is retained in the nucleus 9 . These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

Equine myotonic dystrophy (eMD) is a rare neuromuscular disorder of undetermined origin marked by muscle hypertrophy and stiffness, dystrophic muscle histopathology, and myotonic discharges. In humans, myotonic dystrophy (DM) arises from trinucleotide repeat expansions in dystrophia myotonica protein kinase ( DMPK ) (DM1) or tetranucleotide expansions in cellular nucleic acid-binding protein ( CNBP ) (DM2), which disrupt mRNA processing and induce embryonic splicing patterns across multiple genes. In 6 eMD Quarter Horse types, (2–36 months-of-age) and 8 control Quarter Horses we determined: (1) fiber type composition of triceps , gluteal, and semimembranosus muscles; (2) differential gene (DEG) and protein (DEP) expression using transcriptomic and proteomic analyses; (3) presence of repeat expansions in transcripts of DMPK or CNBP and (4) exon 7 retention in CLCN1 or exon 22 splicing in ATP2A1 . Predominance and clustering of type 1 fibers, expression of embryonic myosin, and upregulated mitochondrial and sarcomeric DEPs characterized eMD hindlimb musculature. Gene ontology (GO) analysis of 730 upregulated DEGs identified numerous GO terms related to morphogenesis of mesoderm-derived tissues and upregulated genes impacting myoD expression in eMD muscle. Top upregulated DEG involved myogenesis ( MYOZ2, SBK2, SBK3, PAMR1 ), neurons, transcription/translation, cytoskeleton, basement/plasma membranes, and calcium binding/transport. Top upregulated proteins also impacted muscle morphogenesis (MUSTN1, CSRP3, TMSBX4, PDLIM, CALD1) as well as categories of mitochondria, sarcomere, extracellular matrix/ basement membrane, transcription, translation, cell cycle regulation, neurons amongst others. Downregulated DEP primarily impacted mitochondria, the sarcomere and glycogen metabolism. Notably, unlike human myotonic dystrophy, trinucleotide repeat expansions were not found in the DMPK 3’UTR (CTG) n nor tetranucleotide repeat expansions (CCTG) n in intron 1 of CNBP . Isoforms of CLCN1 containing fetal exon 7 were detected in equal frequency in eMD and control muscle and exon 22 was not alternatively spliced in ATP2A1 as has been found in DM1. Thus, distinct from DM1 and DM2, eMD is driven by unique molecular mechanisms impacting skeletal muscle morphogenesis, neurons and regulation of gene transcription/translation that alter fiber type composition, distribution and morphology. The origin of myotonia does not appear to be driven by a mutation in CLCN1 or retention of exon CLCN 7. Expanded splice site analysis and further research is warranted to elucidate the cause of myotonia and the distinct etiology of eMD.

Myotonic dystrophy is associated with dysphagia, which can lead to severe complications such as aspiration pneumonia and choking. However, few histopathological studies on dysphagia in myotonic dystrophy have been conducted. In this study, we aimed to validate the FVB/N-Tg(HSA*LR)20bCath mice for studying dysphagia associated with myotonic dystrophy, using videofluoroscopic swallowing study, histological analysis, and immunofluorescence analysis. Videofluoroscopic swallowing study, revealed significant abnormalities during the pharyngeal swallowing phase of swallowing in HSA LR20b mice, including increased pharyngeal residue area and prolonged pharyngeal transit time, suggesting that this mouse model was a valuable tool for studying dysphagia in myotonic dystrophy. These findings might represent a characteristic swallowing pattern in myotonic dystrophy. Histological analysis demonstrated marked variability in muscle fiber size and a high frequency of central nuclei. Additionally, decreased expression of chloride channel 1 was observed in the masseter muscle, suggesting the presence of myotonia. Collectively, these findings provide a foundation for further research into the complex mechanisms underlying myotonic dystrophy associated dysphagia and may inform the development of future treatment strategies.