Ameliorated cellular hallmarks of myotonic dystrophy in hybrid myotubes from patient and unaffected donor cells.

Background: Cell-based strategies are being explored as a therapeutic option for muscular dystrophies, using a variety of cell types from different origin and with different characteristics. Primary pericytes are multifunctional cells found in the capillary bed that exhibit stem cell-like and myogenic regenerative properties. This unique combination allows them to be applied systemically, presenting a promising opportunity for body-wide muscle regeneration.

Differentiation shifts from a reversible to an irreversible heterochromatin state at the DM1 locus.

Epigenetic defects caused by hereditary or de novo mutations are implicated in various human diseases. It remains uncertain whether correcting the underlying mutation can reverse these defects in patient cells. Here we show by the analysis of myotonic dystrophy type 1 (DM1)-related locus that in mutant human embryonic stem cells (hESCs), DNA methylation and H3K9me3 enrichments are completely abolished by repeat excision (CTG2000 expansion), whereas in patient myoblasts (CTG2600 expansion), repeat deletion fails to do so.

Block or degrade? Balancing on- and off-target effects of antisense strategies against transcripts with expanded triplet repeats in DM1.

Antisense oligonucleotide (ASO) therapies for myotonic dystrophy type 1 (DM1) are based on elimination of transcripts containing an expanded repeat or inhibition of sequestration of RNA-binding proteins. This activity is achievable by both degradation of expanded transcripts and steric hindrance, although it is unknown which approach is superior. We compared blocking ASOs with RNase H-recruiting gapmers of equivalent chemistries.

A comprehensive atlas of fetal splicing patterns in the brain of adult myotonic dystrophy type 1 patients.

In patients with myotonic dystrophy type 1 (DM1), dysregulation of RNA-binding proteins like MBNL and CELF1 leads to alternative splicing of exons and is thought to induce a return to fetal splicing patterns in adult tissues, including the central nervous system (CNS). To comprehensively evaluate this, we created an atlas of developmentally regulated splicing patterns in the frontal cortex of healthy individuals and DM1 patients, by combining RNA-seq data from BrainSpan, GTEx and DM1 patients. Thirty-four splice events displayed an inclusion pattern in DM1 patients that is typical for the fetal situation in healthy individuals.

Imaging of CPP Delivery Mechanisms of Oligonucleotides.

Cationic cell-penetrating peptides spontaneously associate with negatively charged oligonucleotides to form submicron nanoparticles, so-called polyplexes. Contact with cells leads to endosomal uptake of these nanoparticles. Oligonucleotide activity critically depends on endosomal release and finally dissociation of polyplexes.

Systemic cell therapy for muscular dystrophies : The ultimate transplantable muscle progenitor cell and current challenges for clinical efficacy.

The intrinsic regenerative capacity of skeletal muscle makes it an excellent target for cell therapy. However, the potential of muscle tissue to renew is typically exhausted and insufficient in muscular dystrophies (MDs), a large group of heterogeneous genetic disorders showing progressive loss of skeletal muscle fibers. Cell therapy for MDs has to rely on suppletion with donor cells with high myogenic regenerative capacity.

Recovery in the Myogenic Program of Congenital Myotonic Dystrophy Myoblasts after Excision of the Expanded (CTG) Repeat.

The congenital form of myotonic dystrophy type 1 (cDM) is caused by the large-scale expansion of a (CTG•CAG) repeat in and . The production of toxic transcripts with long trinucleotide tracts from these genes results in impairment of the myogenic differentiation capacity as cDM's most prominent morpho-phenotypic hallmark. In the current in vitro study, we compared the early differentiation programs of isogenic cDM myoblasts with and without a (CTG)2600 repeat obtained by gene editing.

Advanced Fluorescence Imaging to Distinguish Between Intracellular Fractions of Antisense Oligonucleotides.

Antisense oligonucleotides (AON) have been intensively studied as tools in molecular cell biology and as novel therapeutics in various diseases over the past two decades. Especially cellular uptake and endosomal release of AONs are topics of interest, as these are crucial steps in reaching the subcellular AON target sites and achieving biological activity. We used cell-penetrating peptides (CPPs) to enhance uptake and endosomal release of AONs, and monitored these two processes and the subsequent fate of the AONs by advanced fluorescence microscopy in living cells.

Intrinsic Myogenic Potential of Skeletal Muscle-Derived Pericytes from Patients with Myotonic Dystrophy Type 1.

Pericytes are multipotent, vessel-associated progenitors that exhibit high proliferative capacity, can cross the blood-muscle barrier, and have the ability to home to muscle tissue and contribute to myogenesis. Consequently, pericyte-based therapies hold great promise for muscular dystrophies. A complex multi-system disorder exhibiting muscular dystrophy for which pericytes might be a valuable cell source is myotonic dystrophy type 1 (DM1).

In Vitro Synthesis and RNA Structure Probing of CUG Triplet Repeat RNA.

Aberrant RNA structure plays a central role in the molecular mechanisms guided by repeat RNAs in diseases like myotonic dystrophy (DM), C9orf72-linked amyotrophic lateral sclerosis (ALS) and fragile X tremor/ataxia syndrome (FXTAS). Much knowledge remains to be gained about how these repeat-expanded transcripts are actually folded, especially regarding the properties specific to very long and interrupted repeats. RNA structure can be interrogated by chemical as well as enzymatic probes.

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.

CRISPR/Cas technology holds promise for the development of therapies to treat inherited diseases. Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder with a variable multisystemic character for which no cure is yet available. Here, we review CRISPR/Cas-mediated approaches that target the unstable (CTG•CAG)n repeat in the / gene pair, the autosomal dominant mutation that causes DM1.

The nuclear concentration required for antisense oligonucleotide activity in myotonic dystrophy cells.

Antisense oligonucleotides (ASOs) are a promising class of therapeutics that are starting to emerge in the clinic. Determination of intracellular concentrations required for biologic effects and identification of effective delivery vehicles are crucial for understanding the mode of action and required dosing. Here, we investigated which nuclear oligonucleotide concentration is needed for a therapeutic effect for a triplet repeat-targeting ASO in a muscle cell model of myotonic dystrophy type 1 (DM1).

Certainty-based marking in a formative assessment improves student course appreciation but not summative examination scores.

Background: Study motivation and knowledge retention benefit from regular student self-assessments. Inclusion of certainty-based learning (CBL) in computer-assisted formative tests may further enhance this by enabling students to identify whether they are uninformed or misinformed regarding the topics tested, which may trigger future study actions including instructor consultation.

Methods: Using a cross-over study design involving two out of thirteen computer-assisted formative assessments (CAFAs) of a first-year cell biology course, we compared student-instructor interactions, student learning experiences and final exam scores between two (bio)medical science student cohorts who worked with different CBL-containing CAFAs.

(CTG)n repeat-mediated dysregulation of MBNL1 and MBNL2 expression during myogenesis in DM1 occurs already at the myoblast stage.

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disorder caused by the expression of trinucleotide repeat-containing DMPK transcripts. Abnormally expanded (CUG)n repeats in these transcripts form hairpin-like structures that cause the RNA to accumulate in the cell nucleus by sequestering isoforms of the Muscleblind (MBNL) family, tissue-specific regulators of developmentally programmed, post-transcriptional processes in RNA metabolism. Through this mechanism, the function of MBNL in RNA processing becomes dominantly perturbed, which eventually leads to aberrant alternative splicing and the expression of foetal splice variants of a wide variety of proteins, including the MBNL isoforms themselves.

Expanded CUG repeats in transcripts adopt diverse hairpin conformations without influencing the structure of the flanking sequences.

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disorder caused by expansion of a CTG repeat in the 3'-untranslated region (UTR) of the gene. Mutant transcripts form aberrant structures and anomalously associate with RNA-binding proteins (RBPs). As a first step toward better understanding of the involvement of abnormal mRNA folding in DM1 manifestation, we used SHAPE, DMS, CMCT, and RNase T1 structure probing in vitro for modeling of the topology of the 3'-UTR with normal and pathogenic repeat lengths of up to 197 CUG triplets.

Abnormalities in Skeletal Muscle Myogenesis, Growth, and Regeneration in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) and 2 (DM2) are autosomal dominant degenerative neuromuscular disorders characterized by progressive skeletal muscle weakness, atrophy, and myotonia with progeroid features. Although both DM1 and DM2 are characterized by skeletal muscle dysfunction and also share other clinical features, the diseases differ in the muscle groups that are affected. In DM1, distal muscles are mainly affected, whereas in DM2 problems are mostly found in proximal muscles.

Assisted delivery of antisense therapeutics in animal models of heritable neurodegenerative and neuromuscular disorders: a systematic review and meta-analysis.

Antisense oligonucleotide (AON)-based therapies hold promise for a range of neurodegenerative and neuromuscular diseases and have shown benefit in animal models and patients. Success in the clinic is nevertheless still limited, due to unfavourable biodistribution and poor cellular uptake of AONs. Extensive research is currently being conducted into the formulation of AONs to improve delivery, but thus far there is no consensus on which of those strategies will be the most effective.

Trinucleotide-repeat expanded and normal DMPK transcripts contain unusually long poly(A) tails despite differential nuclear residence.

In yeast and higher eukaryotes nuclear retention of transcripts may serve in control over RNA decay, nucleocytoplasmic transport and premature cytoplasmic appearance of mRNAs. Hyperadenylation of RNA is known to be associated with nuclear retention, but the cause-consequence relationship between hyperadenylation and regulation of RNA nuclear export is still unclear. We compared polyadenylation status between normal and expanded DMPK transcripts in muscle cells and tissues derived from unaffected individuals and patients with myotonic dystrophy type 1 (DM1).

Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood.

CRISPR/Cas9-Induced (CTG⋅CAG) Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing.

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5' or 3' unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair.

Antisense transcription of the myotonic dystrophy locus yields low-abundant RNAs with and without (CAG)n repeat.

The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3' UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown.

A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle.

Muscular manifestation of myotonic dystrophy type 1 (DM1), a common inheritable degenerative multisystem disorder, is mainly caused by expression of RNA from a (CTG·CAG)n-expanded DM1 locus. Here, we report on comparative profiling of expression of normal and expanded endogenous or transgenic transcripts in skeletal muscle cells and biopsies from DM1 mouse models and patients in order to help us in understanding the role of this RNA-mediated toxicity. In tissue of HSA(LR) mice, the most intensely used 'muscle-only' model in the DM1 field, RNA from the α-actin (CTG)250 transgene was at least 1000-fold more abundant than that from the Dmpk gene, or the DMPK gene in humans.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins.

Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2'-O-methyl (2'-OMe) phosphorothioate (PT)-modified (CAG)7 oligo (PS58), which selectively silences mutant DMPK transcripts through recognition of the abnormally long (CUG)n tract.