Natural Compound Boldine Lessens Myotonic Dystrophy Type 1 Phenotypes in DM1 Drosophila Models, Patient-Derived Cell Lines, and HSALR Mice.

Myotonic dystrophy type 1 (DM1) is a complex rare disorder characterized by progressive muscle dysfunction, involving weakness, myotonia, and wasting, but also exhibiting additional clinical signs in multiple organs and systems. Central dysregulation, caused by an expansion of a CTG trinucleotide repeat in the DMPK gene's 3' UTR, has led to exploring various therapeutic approaches in recent years, a few of which are currently under clinical trial. However, no effective disease-modifying treatments are available yet. In this study, we demonstrate that treatments with boldine, a natural alkaloid identified in a large-scale Drosophila-based pharmacological screening, was able to modify disease phenotypes in several DM1 models. The most significant effects include consistent reduction in nuclear RNA foci, a dynamic molecular hallmark of the disease, and noteworthy anti-myotonic activity. These results position boldine as an attractive new candidate for therapy development in DM1.

The myotonic dystrophy type 1 drug development pipeline: 2022 edition.

The beginning of the 20th decade has witnessed an increase in drug development programs for myotonic dystrophy type 1 (DM1). We have collected nearly 20 candidate drugs with accomplished preclinical and clinical phases, updating our previous drug development pipeline review with new entries and relevant milestones for pre-existing candidates. Three interventional first-in-human clinical trials got underway with distinct drug classes, namely AOC 1001 and DYNE-101 nucleic acid-based therapies, and the small molecule pitolisant, which joins the race toward market authorization with other repurposed drugs, including tideglusib, metformin, or mexiletine, already in clinical evaluation. Furthermore, newly disclosed promising preclinical data for several additional nucleic-acid therapeutic candidates and a CRISPR-based approach, as well as the advent into the pipeline of novel therapeutic programs, increase the plausibility of success in the demanding task of providing valid treatments to patients with DM1.

Deciphering the Complex Molecular Pathogenesis of Myotonic Dystrophy Type 1 through Omics Studies.

Omics studies are crucial to improve our understanding of myotonic dystrophy type 1 (DM1), the most common muscular dystrophy in adults. Employing tissue samples and cell lines derived from patients and animal models, omics approaches have revealed the myriad alterations in gene and microRNA expression, alternative splicing, 3' polyadenylation, CpG methylation, and proteins levels, among others, that contribute to this complex multisystem disease. In addition, omics characterization of drug candidate treatment experiments provides crucial insight into the degree of therapeutic rescue and off-target effects that can be achieved. Finally, several innovative technologies such as single-cell sequencing and artificial intelligence will have a significant impact on future DM1 research.

Myotonic dystrophy type 1 drug development: A pipeline toward the market.

Myotonic dystrophy type 1 (DM1) is a multisystemic neuromuscular genetic disease with an estimated prevalence of approximately at least half a million individuals based on its vast ethnic variation. Building upon a well-known physiopathology and several proof-of-concept therapeutic approaches, herein we compile a comprehensive overview of the most recent drug development programs under preclinical and clinical evaluation. Specifically, close to two dozen drug developments, eight of which are already in clinical trials, explore a diversity of new chemical entities, drug repurposing, oligonucleotide, and gene therapy-based approaches. Of these, repurposing of tideglusib, mexiletine, or metformin appear to be therapies with the most potential to receive marketing authorization for DM1.

MicroRNA-Based Therapeutic Perspectives in Myotonic Dystrophy.

Myotonic dystrophy involves two types of chronically debilitating rare neuromuscular diseases: type 1 (DM1) and type 2 (DM2). Both share similarities in molecular cause, clinical signs, and symptoms with DM2 patients usually displaying milder phenotypes. It is well documented that key clinical symptoms in DM are associated with a strong mis-regulation of RNA metabolism observed in patient's cells. This mis-regulation is triggered by two leading DM-linked events: the sequestration of Muscleblind-like proteins (MBNL) and the mis-regulation of the CUGBP RNA-Binding Protein Elav-Like Family Member 1 (CELF1) that cause significant alterations to their important functions in RNA processing. It has been suggested that DM1 may be treatable through endogenous modulation of the expression of MBNL and CELF1 proteins. In this study, we analyzed the recent identification of the involvement of microRNA (miRNA) molecules in DM and focus on the modulation of these miRNAs to therapeutically restore normal MBNL or CELF1 function. We also discuss additional prospective miRNA targets, the use of miRNAs as disease biomarkers, and additional promising miRNA-based and miRNA-targeting drug development strategies. This review provides a unifying overview of the dispersed data on miRNA available in the context of DM.

(CCUG)n RNA toxicity in a Drosophila model of myotonic dystrophy type 2 (DM2) activates apoptosis.

The myotonic dystrophies are prototypic toxic RNA gain-of-function diseases. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by different unstable, noncoding microsatellite repeat expansions - (CTG)DM1 in DMPK and (CCTG)DM2 in CNBP Although transcription of mutant repeats into (CUG)DM1 or (CCUG)DM2 appears to be necessary and sufficient to cause disease, their pathomechanisms remain incompletely understood. To study the mechanisms of (CCUG)DM2 toxicity and develop a convenient model for drug screening, we generated a transgenic DM2 model in the fruit fly Drosophila melanogaster with (CCUG)n repeats of variable length (n=16 and 106). Expression of noncoding (CCUG)106, but not (CCUG)16, in muscle and retinal cells led to the formation of ribonuclear foci and mis-splicing of genes implicated in DM pathology. Mis-splicing could be rescued by co-expression of human MBNL1, but not by CUGBP1 (CELF1) complementation. Flies with (CCUG)106 displayed strong disruption of external eye morphology and of the underlying retina. Furthermore, expression of (CCUG)106 in developing retinae caused a strong apoptotic response. Inhibition of apoptosis rescued the retinal disruption in (CCUG)106 flies. Finally, we tested two chemical compounds that have shown therapeutic potential in DM1 models. Whereas treatment of (CCUG)106 flies with pentamidine had no effect, treatment with a PKR inhibitor blocked both the formation of RNA foci and apoptosis in retinae of (CCUG)106 flies. Our data indicate that expression of expanded (CCUG)DM2 repeats is toxic, causing inappropriate cell death in affected fly eyes. Our Drosophila DM2 model might provide a convenient tool for in vivo drug screening.

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.

CTG repeat expansions in DMPK cause myotonic dystrophy (DM1) with a continuum of severity and ages of onset. Congenital DM1 (CDM1), the most severe form, presents distinct clinical features, large expansions, and almost exclusive maternal transmission. The correlation between CDM1 and expansion size is not absolute, suggesting contributions of other factors. We determined CpG methylation flanking the CTG repeat in 79 blood samples from 20 CDM1-affected individuals; 21, 27, and 11 individuals with DM1 but not CDM1 (henceforth non-CDM1) with maternal, paternal, and unknown inheritance; and collections of maternally and paternally derived chorionic villus samples (7 CVSs) and human embryonic stem cells (4 hESCs). All but two CDM1-affected individuals showed high levels of methylation upstream and downstream of the repeat, greater than non-CDM1 individuals (p = 7.04958 × 10-12). Most non-CDM1 individuals were devoid of methylation, where one in six showed downstream methylation. Only two non-CDM1 individuals showed upstream methylation, and these were maternally derived childhood onset, suggesting a continuum of methylation with age of onset. Only maternally derived hESCs and CVSs showed upstream methylation. In contrast, paternally derived samples (27 blood samples, 3 CVSs, and 2 hESCs) never showed upstream methylation. CTG tract length did not strictly correlate with CDM1 or methylation. Thus, methylation patterns flanking the CTG repeat are stronger indicators of CDM1 than repeat size. Spermatogonia with upstream methylation may not survive due to methylation-induced reduced expression of the adjacent SIX5, thereby protecting DM1-affected fathers from having CDM1-affected children. Thus, DMPK methylation may account for the maternal bias for CDM1 transmission, larger maternal CTG expansions, age of onset, and clinical continuum, and may serve as a diagnostic indicator.

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3' untranslated region of the DMPK gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, miR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652 all failed to show consistent differences in serum expression in subsequent validation experiments.

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1.

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1-MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.

In vivo strategies for drug discovery in myotonic dystrophy disorders.

Myotonic dystrophy (DM) is a complex neuromuscular genetic disease for which there is currently no valid therapy. The recent development of non-mammal animal models opened up the possibility of performing drug discovery in vivo, using as screening readout phenotypes with underlying molecular parallels to the disease. In this review we discuss the state of the art technologies already used in large scale drug screening and provide guidance for further development of novel technologies.

Replacement of the myotonic dystrophy type 1 CTG repeat with 'non-CTG repeat' insertions in specific tissues.

BACKGROUND: Recently, curious mutations have been reported to occur within the (CTG)n repeat tract of the myotonic dystrophy type 1 (DM1) locus. For example, the repeat, long presumed to be a pure repeat sequence, has now been revealed to often contain interruption motifs in a proportion of cases with expansions. Similarly, a few de novo somatic CTG expansions have been reported to arise from non-expanded DM1 alleles with 5-37 units, thought to be genetically stable. AIMS AND METHODS: This study has characterised a novel mutation configuration at the DM1 CTG repeat that arose as somatic mosaicism in a juvenile onset DM1 patient with a non-expanded allele of (CTG)12 and tissue specific expansions ranging from (CTG)1100 to 6000. RESULTS: The mutation configuration replaced the CTG tract with a non-CTG repeat insertion of 43 or 60 nucleotides, precisely placed in the position of the CTG tract with proper flanking sequences. The inserts appeared to arise from a longer human sequence on chromosome 4q12, and may have arisen through DNA structure mediated somatic inter-gene recombination or replication/repair template switching errors. De novo insertions were detected in cerebral cortex and skeletal muscle, but not in heart or liver. Repeat tracts with -1 or -2 CTG units were also detected in cerebellum, which may have arisen by contractions of the short (CTG)12 allele. CONCLUSION: This non-CTG configuration expands current understanding of the sequence variations that can arise at this hypermutable site.

Maternal germline-specific effect of DNA ligase I on CTG/CAG instability.

The instability of (CTG)•(CAG) repeats can cause >15 diseases including myotonic dystrophy, DM1. Instability can arise during DNA replication, repair or recombination, where sealing of nicks by DNA ligase I (LIGI) is a final step. The role of LIGI in CTG/CAG instability was determined using in vitro and in vivo approaches. Cell extracts from a human (46BR) harbouring a deficient LIGI (∼3% normal activity) were used to replicate CTG/CAG repeats; and DM1 mice with >300 CTG repeats were crossed with mice harbouring the 46BR LigI. In mice, the defective LigI reduced the frequency of CTG expansions and increased CTG contraction frequencies on female transmissions. Neither male transmissions nor somatic CTG instability was affected by the 46BR LigI - indicating a post-female germline segregation event. Replication-mediated instability was affected by the 46BR LIGI in a manner that depended upon the location of Okazaki fragment initiation relative to the repeat tract; on certain templates, the expansion bias was unaltered by the mutant LIGI, similar to paternal transmissions and somatic tissues; however, a replication fork-shift reduced expansions and increased contractions, similar to maternal transmissions. The presence of contractions in oocytes suggests that the DM1 replication profile specific to pre-meiotic oogenesis replication of maternal alleles is distinct from that occurring in other tissues and, when mediated by the mutant LigI, is predisposed to CTG contractions. Thus, unlike other DNA metabolizing enzymes studied to date, LigI has a highly specific role in CTG repeat maintenance in the maternal germline, involved in mediating CTG expansions and in the avoidance of maternal CTG contractions.

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites, including expanded (CAG)n/(CTG)n repeats.

Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu 4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83•(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500•(CTG)500 or (CAG)800•(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n•(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.

Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues.

Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus.

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

Repeat instability as the basis for human diseases and as a potential target for therapy.

Expansions of repetitive DNA sequences cause numerous human neurological and neuromuscular diseases. Ongoing repeat expansions in patients can exacerbate disease progression and severity. As pathogenesis is connected to repeat length, a potential therapeutic avenue is to modulate disease by manipulating repeat expansion size--targeting DNA, the root-cause of symptoms. How repeat instability is mediated by DNA replication, repair, recombination, transcription and epigenetics may explain its contribution to pathogenesis and give insights into therapeutic strategies to block expansions or induce contractions.

CTG/CAG repeat instability is modulated by the levels of human DNA ligase I and its interaction with proliferating cell nuclear antigen: a distinction between replication and slipped-DNA repair.

Mechanisms contributing to disease-associated trinucleotide repeat instability are poorly understood. DNA ligation is an essential step common to replication and repair, both potential sources of repeat instability. Using derivatives of DNA ligase I (hLigI)-deficient human cells (46BR.1G1), we assessed the effect of hLigI activity, overexpression, and its interaction with proliferating cell nuclear antigen (PCNA) upon the ability to replicate and repair trinucleotide repeats. Compared with LigI(+/+), replication progression through repeats was poor, and repair tracts were broadened beyond the slipped-repeat for all mutant extracts. Increased repeat instability was linked only to hLigI overexpression and expression of a mutant hLigI incapable of interacting with PCNA. The endogenous mutant version of hLigI with reduced ligation activity did not alter instability. We distinguished the DNA processes through which hLigI contributes to trinucleotide instability. The highest levels of repeat instability were observed under the hLigI overexpression and were linked to reduced slipped-DNAs repair efficiencies. Therefore, the replication-mediated instability can partly be attributed to errors during replication but also to the poor repair of slipped-DNAs formed during this process. However, repair efficiencies were unaffected by expression of a PCNA interaction mutant of hLigI, limiting this instability to the replication process. The addition of purified proteins suggests that disruption of LigI and PCNA interactions influences trinucleotide repeat instability. The variable levels of age- and tissue-specific trinucleotide repeat instability observed in myotonic dystrophy patients and transgenic mice may be influenced by varying steady state levels of DNA ligase I in these tissues and during different developmental windows.