BACKGROUND:Myotonic dystrophy type 1 (DM1) is a multisystem disorder with autosomal dominant inheritance, caused by the abnormal expansion of the CTG triplet in the DMPK gene. Biomarker discovery in DM1 is crucial for monitoring disease progression. METHODS:We performed RNA sequencing on blood, skin and muscle samples from the same patients to assess splicing events. Mis-splicing events were identified using the Mann-Whitney U rank-sum test, and per cent spliced in for exons was correlated with repeat expansion size using Spearman's correlation. We also examined the relationship between mis-splicing and disease severity through Fisher's exact test and correlation analyses. RESULTS:We identified 937, 384 and 1216 mis-splicing events in muscle, blood and skin, respectively. Of these, 52 exons in muscle and 10 in blood correlated with estimated progenitor allele length (false discovery rate (FDR) <0.1), but none in skin. Notably, nine exons in blood correlated with total muscle mis-splicing (FDR<0.05), suggesting their potential as biomarkers of severity. CONCLUSION:This is the first study to identify splicing dysregulation in blood and skin in patients with DM1 and identify novel potential blood-based mis-splicing biomarkers for disease severity. The correlation between several blood exons and the muscle splicing dysregulation indicates that blood-based biomarkers can be valuable for assessing disease severity, monitoring disease progression and evaluating treatment efficacy. Larger sample sizes may be necessary to clarify the relationship between mis-splicing and disease severity.

ABSTRACT Background The restoration of uniformly distributed dystrophin protein expression is an important consideration for the development of advanced therapeutics for Duchenne muscular dystrophy (DMD). Methods We have generated a novel genetic mouse model (mdx52‐XistΔhs) that expresses variable and nonuniformly distributed dystrophin protein from birth as a consequence of skewed X‐chromosome inactivation. mdx52‐XistΔhs myofibers are heterokaryons containing a mixture of myonuclei expressing either wild‐type or mutant dystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being spatially restricted to corresponding dystrophin‐expressing myonuclear domains. This phenotype models the situation in female dystrophinopathy and dystrophic muscle in which dystrophin has been incompletely restored by partially effective experimental therapeutics. Dystrophin distribution was assessed in mdx52‐XistΔhs muscle sections and isolated single myofibers by immunostaining and RNA‐FISH analysis. Results Total dystrophin expression increased by ~2.8‐fold (p < 0.010) in aged (60‐week‐old) mdx52‐XistΔhs mice relative to 6‐week‐old adults, suggestive of an aging‐associated accumulation of dystrophin‐expressing myonuclei through positive selection, although this was insufficient to resolve sarcolemmal dystrophin patchiness. Nonuniformly distributed dystrophin conferred partial protection against pathology‐related muscle turnover in an expression‐level‐dependent manner in both adult and aged mdx52‐XistΔhs mice compared to mice expressing no dystrophin. Isolated mdx52‐XistΔhs myofibers exhibited patchy ‘zebra‐like’ banding of dystrophin sarcolemmal coverage that colocalized with β‐dystroglycan but not neuronal nitric oxide synthase, which was uniformly distributed. Systematic classification of isolated mdx52‐XistΔhs myofibers revealed unexpected and profound differences associated with central nucleation, with dystrophin found to be absent in centrally nucleated myofibers and myofiber segments. Muscle injury alone was insufficient to recapitulate this phenomenon, suggesting that it is a feature of the dystrophic environment. Dmd mRNA was found to be present throughout centrally nucleated segments, and proteins such as titin and F‐actin were uniformly distributed, suggesting that dystrophin is specifically repressed at the protein level in these regions. The microtubule network was moderately disrupted in mdx52‐XistΔhs ‘zebra‐banded’ fibers, but this effect was not different between dystrophin‐positive and dystrophin‐negative myofiber subdomains. By contrast, centrally nucleated mdx52‐XistΔhs myofibers exhibited severe microtubule network disruption. Conclusions These findings reveal new insights into the importance of dystrophin spatial localization and identify a previously unappreciated barrier to effective therapeutic dystrophin restoration, particularly within regenerated or centrally nucleated myofibers.

Myotonic dystrophy type 1 (DM1) is a multisystemic autosomal dominant disorder caused by the expansion of an unstable CTG•CAG repeat in the DMPK gene. This study examined whether differential expression of DNA repair genes in three different tissues from the same DM1 patient contributed to tissue-specific somatic instability of the repeat tract. RNA-Seq was used to quantify expression levels of eight DNA repair genes (MSH2, MSH3, MSH6, MLH1, MLH3, PMS2, LIG1, and FAN1). Results indicate that the expression levels varied significantly across tissues, with no inter-tissue correlations, suggesting independent regulation and patient-specific differences. Somatic expansion of the repeat tract in blood and muscle was effectively predicted by a complex ePALxAge interaction, while in muscle it was further influenced by MSH3 and PMS2 gene expression, confirming their role as tissue-specific genetic modifiers in DM1. Although only marginally significant, muscle expression of PMS2 and FAN1 appeared to affect age-at-onset: higher FAN1 expression was associated with reduced somatic expansion and later onset. Our findings also suggest a complex competitive balance between promoters and stabilizers of repeat instability, shaping muscle expansion dynamics and potentially modifying clinical onset. Overall, these results indicate that certain DNA repair genes exert stronger, tissue-dependent effects on somatic instability. We confirm that MSH3, PMS2, and FAN1 act as key modifiers not only of somatic expansion, especially in skeletal muscle, but also of DM1 severity. Although RT-qPCR data might be required to validate some of these results, these genes therefore represent promising therapeutic targets for modulating disease progression.

Spinocerebellar ataxia type 8 (SCA8) is a member of a group of dominantly inherited, debilitating neurological diseases caused by CAG•CTG expansions for which there are no effective treatments. RAN translation, which was discovered in SCA8, has previously been shown to occur across CAG and CUG expansion transcripts, making treatments for SCA8 potentially relevant to a broad group of diseases, including SCA1, SCA2, SCA3, SCA6, SCA7, SCA12, Huntington's disease, and myotonic dystrophy type 1. In addition, CUG and CAG expansion transcripts have been reported to cause RNA gain-of-function effects. Using SCA8 BAC transgenic mice as a model for CAG•CTG expansion diseases, we now show that metformin improves ambulatory performance using rotarod, DigiGait, and open-field testing. At the molecular level, metformin-treated mice show reduced RAN protein levels and improved splicing, without altering sense or antisense RNA levels. Metformin-treated mice also show decreased neuroinflammation, with reduced astrogliosis and fewer activated microglia. These data provide strong preclinical support for testing metformin in clinical trials for SCA8 and potentially the broader group of CAG•CTG repeat expansion disorders.

Muscleblind-like (MBNL) RNA-binding proteins (RBPs) possess modular domains that mediate regulation of alternative splicing and RNA localization. In Myotonic Dystrophy Type 1, a CTG repeat expansion disorder, MBNL is sequestered into intranuclear RNA foci, impairing its function. Previous studies found that MBNL self-associates through its exon 7, but the nature of this interaction is not well understood. We identified a cysteine in MBNL1 exon 7 that enables dimerization through the formation of an intermolecular disulfide bond. We likewise demonstrate that MBNL2 dimerizes by forming disulfide bonds between multiple cysteines in its carboxy-terminus. Nucleocytoplasmic fractionation revealed a greater proportion of MBNL1 dimer in the nucleus, suggesting a nuclear function for the MBNL1 dimer. We investigated a connection between MBNL1 dimerization and MBNL1-mediated regulation of alternative splicing. To accomplish this, we mutated the MBNL1 cysteine in question to alanine (C325A) and performed RNAseq. We uncovered novel splicing events sensitive to MBNL1 dimerization. We also found that MBNL1 C325A, when co-expressed with expanded CTG repeats, produces smaller, more numerous foci, suggesting a role for the MBNL1 dimer in maintaining foci integrity. These results provide insight into biological and pathological mechanisms of MBNL1 dimerization and suggest that other RBPs might similarly dimerize to regulate function.

Steric-blocking antisense oligonucleotides rescue myotonic dystrophy type 1 phenotypes in preclinical models and are under evaluation in clinical trials. However, the 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 designed to block pathogenic CUG repeat expansions in HSALR mice. A single systemic administration rescued mis-splicing and eliminated myotonia 1 week post-injection, with partial splicing rescue evident after 24 h. Interestingly, some exons showed more robust rescue than others, but the relationship between muscleblind-like (MBNL) protein concentration ([MBNL]) and percent spliced in (Ψ) did not fully explain the extent of rescue. We hypothesized that since pre-existing transcripts must be degraded to reveal the 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 RNA sequencing (RNA-seq) validated these predictions. Overall, we show that transcript turnover influences Ψ during periods of dynamically changing [MBNL] and recommend considering this when selecting splicing biomarkers and interpreting responses to therapeutic interventions.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder with no approved therapeutics targeting the disease mechanism. DM1 is caused by the expression of expanded CUG repeat RNA (CUGexp), which sequester the muscleblind-like (MBNL) family of RNA binding proteins leading to dysregulated alternative splicing and a host of downstream impacts. While previous studies showed that diamidines rescued DM1 dysregulated alternative splicing events, their potential was limited by toxicity and off-target effects. A new class of modified polycyclic compounds (MPCs), based on diamidines, were created and screened in DM1 patient-derived cell lines. This approach identified MPC03 and MPC04 as being capable of rescuing DM1 dysregulated splicing events at low nanomolar concentrations with no obvious toxicity and limited off-target effects. In a DM1 mouse model, treatment with MPC03 and MPC04 reduced CUGexp RNA levels and partially rescued DM1 mis-splicing. Binding data and modeling showed that lead MPCs bind to CUGexp RNA, and in cells lacking CUG repeats, MPC activity was absent, suggesting that these compounds displace sequestered MBNL proteins from CUGexp RNA. Taken together, MPCs show therapeutic promise across multiple DM1 models.

Spinocerebellar ataxia type 8 is one member of a larger group of dominantly inherited, debilitating neurological diseases caused by CTG*CAG expansions for which there are no effective disease-targeting treatments. RAN translation, which was discovered in SCA8, has previously been shown to occur across CAG and CUG expansion transcripts, making treatments that work for SCA8 potentially relevant to a much broader group of diseases, including SCA1, 2, 3, 6, 7, 12, Huntington’s Disease (HD) Fuch’s endothelial corneal dystrophy (FECD), and myotonic dystrophy type 1 (DM1). The FDA-approved drug, metformin, has been previously shown to reduce RAN protein levels in cells overexpressing SCA8 CAG repeats. Here we show, using SCA8 BAC transgenic mice, that metformin treatment improves ambulatory performance, including rotarod, DigiGait, and open field measures. At the molecular level, metformin-treated mice show reduced RAN protein levels and improved splicing abnormalities without changing the levels of the expanded RNAs. Metformin-treated mice also show decreased neuroinflammation with reduced levels of astrogliosis and reduced numbers of activated microglia. Taken together, these data provide strong support for testing FDA-approved metformin in clinical trials for SCA8 and potentially the broader group of CAG*CTG repeat expansion disorders. One Sentence Summary Metformin improves behavior, neuropathological and molecular phenotypes in SCA8 BAC transgenic mice. ### Competing Interest Statement Drs Ranum and Zu have patents and pending patents on RAN translation

Alzheimer’s disease (AD) affects more than 10% of the population ≥65 y of age, but the underlying biological risks of most AD cases are unclear. We show anti-poly-glycine-arginine (a-polyGR) positive aggregates frequently accumulate in sporadic AD autopsy brains (45/80 cases). We hypothesize that these aggregates are caused by one or more polyGR-encoding repeat expansion mutations. We developed a CRISPR/deactivated-Cas9 enrichment strategy to identify candidate GR-encoding repeat expansion mutations directly from genomic DNA isolated from a-polyGR(+) AD cases. Using this approach, we isolated an interrupted (GGGAGA) n intronic expansion within a SINE-VNTR-Alu element in CASP8 ( CASP8 -GGGAGA EXP ). Immunostaining using a-polyGR and locus-specific C-terminal antibodies demonstrate that the CASP8 -GGGAGA EXP expresses hybrid poly(GR)n(GE)n(RE)n proteins that accumulate in CASP8 -GGGAGA EXP (+) AD brains. In cells, expression of CASP8 -GGGAGA EXP minigenes leads to increased p-Tau (Ser202/Thr205) levels. Consistent with other types of repeat-associated non-AUG (RAN) proteins, poly(GR)n(GE)n(RE)n protein levels are increased by stress. Additionally, levels of these stress-induced proteins are reduced by metformin. Association studies show specific aggregate promoting interrupted CASP8 -GGGAGA EXP sequence variants found in ~3.6% of controls and 7.5% AD cases increase AD risk [ CASP8 -GGGAGA-AD-R1; OR 2.2, 95% CI (1.5185 to 3.1896), P = 3.1 × 10 −5 ]. Cells transfected with a high-risk CASP8 -GGGAGA-AD-R1 variant show increased toxicity and increased levels of poly(GR)n(GE)n(RE)n aggregates. Taken together, these data identify polyGR(+) aggregates as a frequent and unexpected type of brain pathology in AD and CASP8 -GGGAGA-AD-R1 alleles as a relatively common AD risk factor. Taken together, these data support a model in which CASP8 -GGGAGA EXP alleles combined with stress increase AD risk.

Nanoparticle vaccines displaying combinations of SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) could protect against SARS-CoV-2 variants and spillover of zoonotic sarbecoviruses into humans. Using a computational approach, we designed variants of SARS-CoV-2 RBDs and selected 7 natural sarbecovirus RBDs, each predicted to fold properly and abrogate antibody responses to variable epitopes. RBDs were attached to 60-mer nanoparticles to make immunogens displaying two (mosaic-2COMs), five (mosaic-5COM), or seven (mosaic-7COM) different RBDs for comparisons with mosaic-8b, which elicited cross-reactive antibodies and protected animals from sarbecovirus challenges. Naive and COVID-19 pre-vaccinated mice immunized with mosaic-7COM elicited antibodies targeting conserved RBD epitopes, and their sera exhibited higher binding and neutralization titers against sarbecoviruses than mosaic-8b. Mosaic-2COMs and mosaic-5COM elicited higher antibody potencies against some SARS-CoV-2 variants than mosaic-7COM. However, mosaic-7COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons, supporting its use to protect against SARS-CoV-2 variants and zoonotic sarbecoviruses.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader", a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Myotonic dystrophy type 1 (DM1), the leading cause of adult-onset muscular dystrophy, is caused by a CTG repeat expansion. Expression of the repeat causes widespread alternative splicing (AS) defects and downstream pathogenesis, including significant skeletal muscle impacts. The HSA(LR) mouse model plays a significant role in therapeutic development. This mouse model features a transgene composed of approximately 220 interrupted CTG repeats, which results in skeletal muscle pathology that mirrors DM1. To better understand this model and the growing number of therapeutic approaches developed with it, we performed a meta-analysis of publicly available RNA sequencing data for AS changes across three widely examined skeletal muscles: quadriceps, gastrocnemius, and tibialis anterior. Our analysis demonstrated that transgene expression correlated with the extent of splicing dysregulation across these muscles from gastrocnemius (highest), quadriceps (medium), to tibialis anterior (lowest). We identified 95 splicing events consistently dysregulated across all examined datasets. Comparison of splicing rescue across seven therapeutic approaches showed a range of rescue across the 95 splicing events from the three muscle groups. This analysis contributes to our understanding of the HSA(LR) model and the growing number of therapeutic approaches currently in preclinical development for DM1.

RNA fluorescence in situ hybridization (FISH) is a powerful method to determine the abundance and localization of mRNA molecules in cells. While modern RNA FISH techniques allow quantification at single molecule resolution, most methods are optimized for mammalian cell culture and are not easily applied to in vivo tissue settings. Single-molecule RNA detection in skeletal muscle cells has been particularly challenging due to the thickness and high autofluorescence of adult muscle tissue and a lack of in vitro models for mature muscle cells (myofibers). Here, we present a method for isolation of adult myofibers from mouse skeletal muscle and detection of single mRNA molecules and proteins using multiplexed RNA FISH and immunofluorescence.

Pathogenic repeat sequences underlie several human disorders, including amyotrophic lateral sclerosis, Huntington's disease, and myotonic dystrophy. Here, we speak to several researchers about how repeat sequences have been implicated in affecting all aspects of the Central Dogma of molecular biology through their effects on DNA, RNA, and protein.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed diseaseassociated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG)exp, harbored in the 3′ untranslated region (3′ UTR) of dystrophia myotonica protein kinase (DMPK) mRNA and drives disease through a gain-of-function mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG)exp in vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG)exp. The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG)exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.