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Molecular Therapies for Myotonic Dystrophy Type 1
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This entry is adapted from the peer-reviewed paper 10.3390/ijms23094622

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy affecting many different body tissues, predominantly skeletal and cardiac muscles and the central nervous system. The expansion of CTG repeats in the DM1 protein-kinase (DMPK) gene is the genetic cause of the disease. The pathogenetic mechanisms are mainly mediated by the production of a toxic expanded CUG transcript from the DMPK gene. With the availability of new knowledge, disease models, and technical tools, much progress has been made in the discovery of altered pathways and in the potential of therapeutic intervention, making the path to the clinic a closer reality. 

myotonic dystrophy trinucleotide-expansion disease DM1 mice antisense oligonucleotides molecular therapy gene editing

1. Introduction

Myotonic dystrophy type 1 (DM1) is caused by an unstable expanded CTG repeat located within the 3′ untranslated region (3′ UTR) of the DMPK gene. The molecular mechanisms of DM1 are mainly the consequence of accumulation of mutant DMPK transcripts into ribonuclear foci leading to the impairment of alternative splicing and normal gene expression. Cell and animal models of DM1 have been crucial to providing insight into disease mechanisms and to revealing new therapeutic targets. 

2. Myotonic Dystrophy Type 1: Clinical Features and Pathogenetic Mechanisms

DM1 is the most common dystrophy in adults, having an estimated worldwide prevalence of 1:20,000, with a recent report of 4.76:10,000 for DMPK CTG expansion ≥50 CTG repeats in a newborn screening program in New York State, USA [1]. Clinical features of DM1 (Steinert’s disease, OMIM# 160900) include muscle weakness, dysphagia, neuromuscular respiratory insufficiency, cardiac complications and cognitive, intellectual or behavioral impairment as well as sleep disorders. In the most severe forms, life quality and expectancy are seriously compromised [2][3][4]. DM1 results from CTG-repeat expansions in the 3′ UTR of the DMPK gene on chromosome 19 [5][6]. The disease severity and age of onset are broadly correlated with the number of CTG repeats, with the highest (over 750) in the congenital form, while in non-affected individuals the number of repeats is up to 35 [2]. The number of repeats is usually unstable and tends to increase in some body tissues during lifetime (somatic instability) as well as in successive generations, leading to the phenomenon called “anticipation”, where children of DM1 patients have a higher repeat number and more severe phenotypes compared to their parents [7]. Interestingly, in DM1 families with variant repeats, where GGC, CCG and CTC interruptions are present within the CTG-repeat array, the repeats are stabilized and the disease phenotypes are milder [8][9]. Several pathogenic mechanisms likely contribute to disease in DM1 (Figure 1) [4][10]. At the DNA level, epigenetic modifications may impact the development or severity of the phenotype in DM1 patients [11]. In DM1 patient-derived cells and in a DM1 mouse model, the hairpin-like structures of the repeats can induce chromatin changes, such as CpG methylation, resulting in haploinsufficiency of DMPK and neighboring genes, or cause replication-fork stalling during DNA duplication, leading to cell stress [12][13][14]. Large experimental evidence supports the hypothesis of an RNA gain-of-function mechanism of the mutated DMPK transcript. CUG-containing RNAs sequester crucial nuclear factors of the muscleblind-like (MBNL) family into ribonuclear foci, thus preventing their normal functions that are mainly associated with the regulation of alternative splicing [15]. Splicing regulation is required for the proper development and maintenance of tissues in which the DMPK gene is highly expressed, such as in muscle and the nervous system [16]. The MBNL family and the CUGBP Elav-like family (CELF) are among the most important splicing regulators in skeletal and cardiac muscle, and act antagonistically on several pre-mRNA targets [17][18]. Nuclear retention of MBNL proteins in nuclear foci prevents pre-mRNA processing and export to the cytoplasm, leading to a decrease in protein translation, and the loss of functional MBNL1 is accompanied by CELF1 upregulation [19]. The increase in CELF1 levels is induced by protein-kinase-C (PKC)-mediated hyperphosphorylation, leading to protein stabilization [20]. Both sense and antisense repeated RNAs have been shown to contribute to the clinical phenotype of nucleotide-expansion diseases [21]. An antisense transcript emanating from the DMPK-adjacent SIX5 regulatory region spanning the CTG expansion was first identified in DM1 patient-derived cells. The transcript was shown to be converted into siRNAs, which are able to recruit DNA and histone methyltransferases, leading to heterochromatin formation [22]. Interestingly, in transgenic mice carrying the human DMPK locus, in addition to CUG-containing transcripts, CAG-containing transcripts were also found to form distinct ribonuclear foci [23].
Ijms 23 04622 g001 550
Figure 1. DM1 pathogenetic mechanisms and therapeutic strategies. The actions of molecular therapies for DM1 at different pathogenetic levels are illustrated: (1) at DMPK gene, drugs can inhibit CTG-repeat transcription and induce repeat contraction; ZFN, TALEN or CRISPR/Cas9 nucleases can modify gene sequence by inducing CTG-repeat contractions or deletions, or by inserting premature polyadenylation signals; (2) mutated DMPK mRNA can be functionally inactivated by drugs inducing degradation or binding to CUG repeats; (3) MBNL can be released from CUG repeats by disruption of MBNL:CUG interaction through competitive binding, and CELF levels can be regulated by protein kinase C and glycogen synthase kinase 3β; (4) altered signaling pathways downstream of DMPK transcript can be rescued by modulation of splicing and miRNAs; circRNAs and RAN translation could also be targets of future therapies.
Other mechanisms involved in DM1 pathogenesis are repeat-associated non-ATG (RAN) translation (reviewed in [24]), which results in the production of toxic protein aggregates containing polyglutamine from antisense CAG-repeated transcripts [25][26], microRNA (miRNA) dysregulation [27][28][29][30], and upregulation of circular RNA (circRNA) expression [31][32][33]. In addition to the ones described above, other signaling cascades are affected by the toxic DMPK RNA and may play important roles in DM1 pathogenesis. For example, MBNL and CELF regulators, besides being key splicing regulators, are likely involved in cytoplasmic pathogenetic processes altering proteostasis and sarcomere structure (reviewed in [34]). Omics studies have added new information to the previous knowledge, revealing several alterations in gene expression, alternative splicing, CpG methylation and proteins levels that potentially contribute to DM1 pathogenesis. In perspective, these new approaches can be crucial to evaluate the degree of therapeutic rescue and the off-target effects of drug candidates (reviewed in [35]).

3. DM1 Cell and Animal Models

In vitro models of DM1 have greatly contributed to clarifying the pathogenetic mechanisms of the disease. Among these, there are engineered cell lines with CTG repeats of different lengths inserted in minigenes [36][37], DM patient-derived primary myoblasts, immortalized myoblasts or MYOD1-converted fibroblasts [38][39][40][41], and embryonic stem cells [42]. Additionally, induced pluripotent stem cells (iPSCs) and iPSC-derived distinct cell types were used to study tissue-specific DM1 pathological alterations [43][44][45][46]. Recently, the first 3D in vitro human-muscle model of DM1 was developed by encapsulating patient-derived MYOD1-converted fibroblasts in hydrogels [47]. All of these cell models reproduce molecular alterations typical of DM1 and have been very useful for discovering crucial molecules and cellular pathways involved in the disease and for testing therapeutic strategies.
Drosophila models have also been used by several groups to study DM1. Interrupted CTG repeats of various lengths driven by either constitutive or inducible promoters were expressed in flies and DM1-related molecular as well as phenotypic alterations were observed in flies carrying more than 480 repeats [48][49][50]. Although DM1 modeling is complicated by the multifaceted impact of the DM1 mutation, many DM1 mouse models have been generated over time through the silencing of the Dmpk gene or the Mbnl family genes; alternatively, mouse models expressing CELF proteins or toxic CTG repeats in various tissues were produced, in order to mimic the different aspects of the disease and to discover therapeutic molecules (Table 1). It is unclear whether the DMPK haploinsufficiency observed in DM1 patients may affect functions of the tissues in which the gene is normally highly expressed, such as muscles and the central nervous system (CNS). To address this question, different Dmpk-knockout (KO) mouse models have been generated and characterized through the years with different results. Initial reports on Dmpk-KO mice described cardiac conduction defects [51] and mild myopathy [52]. Since these mice models were characterized by a mixed genetic background possibly leading to confounding effects, more recently a Dmpk-KO model backcrossed to two different pure genetic backgrounds was generated. This model did not confirm the previous observations, but showed that Dmpk gene deletion does not compromise cardiac or skeletal-muscle function [53]. Additionally, DMPK transcript silencing through antisense oligonucleotides (ASOs) was well tolerated in mice, rats and monkeys [54]. These findings suggest that reduction in DMPK expression should not be a prominent cause of the disease. Given the crucial regulatory role of MBNL proteins in DM1, different Mbnl KO models were generated to elucidate the role of each MBNL protein in the disease. Mbnl1 and Mbnl2 loss of function resulted in muscular and CNS symptom manifestation, respectively [55][56][57], while Mbnl3 KO caused a progressive delay in muscle regeneration and embryonic muscle differentiation abnormalities, in agreement with their expression profiles during development [58]. Mice with double Mbnl1/Mbnl2 KO or Mbnl1/Mbnl3 KO exhibited more severe phenotypes compared with the single KO [59][60] and the triple Mbnl1/2/3 KO in muscle tissues recapitulated the severe phenotype observed in congenital DM1, in both newborn and adult mice [61], supporting the idea of a prominent role of MBNL proteins and alternative splicing dysregulation in DM1 pathogenesis. To determine the role of CELF proteins in DM1 pathogenesis, mouse models overexpressing CELF1 and CELF2 in skeletal and/or cardiac muscle were generated [62][63][64][65]. Overexpression of CELF1 was shown to reproduce DM1-associated histopathological and functional changes [63]. Notably, CELF1/2 overexpressing mouse models have revealed a strong pattern of antagonistic regulation of mRNA levels by CELF and MBNL proteins through competitive binding to 3′ UTR regions [64] (Table 1A).
Table 1. DM1 mouse models used for studying pathogenetic mechanisms and/or molecular therapies.
Table
(A) Knockout and Overexpressing Models
Mouse Model Generation Strategy DM1-Like Features Limitations Research Application Ref
DMPK-/- Dmpk KO via replacement of 5′-UTR and exons 1-7 with hygromycin cassette Late-onset mild myopathy and altered Ca++ homeostasis Mild phenotype; possible confounding insertional effects on flanking genes; mixed genetic background Relevance of absence of DMPK protein to DM1 phenotype [52][66]
DMPK-/- Dmpk KO via replacement of 5′-UTR and exons 1-7 with neomycin cassette Late-onset mild myopathy; decreased force generation; altered Na+ currents in skeletal muscles; cardiac conduction defects Mild phenotype; possible confounding insertional effects on flanking genes; mixed genetic background Relevance of absence of DMPK protein to DM1 phenotype [51][67]
DMPK-/- Dmpk KO via replacement of 5′-UTR and exons 1-7 with hygromycin cassette No phenotype Failure to replicate the DM1 phenotype Relevance of absence of DMPK protein to DM1 phenotype [53]
Mbnl1ΔE3/ΔE3 Mbnl1 KO via targeted deletion of Mbnl1 exon 3 Mild myotonia and myopathy (centralized nuclei, split fibers); heart conduction defects; progressive cataracts; AS alterations Mild muscle phenotype; mild brain alterations; limited spliceopathy Evaluation of MBNL1 splicing regulation to DM1 phenotype [56][57]
Mbnl2ΔE2/ΔE2 Mbnl2 KO via targeted deletion of Mbnl2 exon 2 Development of several CNS alterations (REM sleep propensity, deficit in spatial memory, decreased synaptic plasticity), AS alterations Failure to replicate the DM1 muscular phenotype Evaluation of MBNL2 splicing regulation to DM1 phenotype [55]
Mbnl3ΔE2 Mbnl3 KO via targeted deletion of Mbnl3 exon 2 (X-linked) Progressive delay in muscle regeneration; abnormalities in embryonic muscle differentiation leading to neonatal hypotonia Possible compensation by MBNL3 truncated isoform or other MBNl family members Evaluation of MBNL3 contribution to DM1 phenotype [58]
Mbnl1ΔE3/ΔE3;
Mbnl2C/C;
Myo-Cre+/-		 Mbnl1 KO; skeletal-muscle specific Cre-mediated Mbnl2 KO Small size at birth and skeletal abnormalities; myopathy and severe motor deficits; AS alterations also in brain tissues High neonatal mortality and reduced lifespan Evaluation of MBNL1 and MBNL2 contribution to DM1 muscular phenotype [60]
Mbnl1ΔE3/ΔE3;
Mbnl3ΔE2		 Mbnl1 and Mbnl3 KO via targeted deletion of Mbnl1 exon 3 and Mbnl3 exon 2 Myotonia and myopathy; reduction in muscle strength; chloride currents alteration; AS alterations; translation defects AS alterations similar to Mbnl1 single knock out; lack of brain alterations Evaluation of MBNL1 and MBNL3 contribution to DM1 phenotype [59]
Mbnl1ΔE3/ΔE3;
Mbnl2C/C;
Mbnl3C;
Myo-Cre+/-		 Mbnl1 KO; muscle-specific Cre-mediated Mbnl2 and Mbnl3 KO Severe congenital myopathy and spliceopathy, severe respiratory difficulties and muscle wasting in adults; gene expression changes High neonatal mortality and reduced lifespan Evaluation of all MBNL proteins loss contribution to DM1 muscular phenotype [61]
MCKCUGBP1 Insertion of human CELF1 transgene under striated-muscle-specific MCK mouse promoter Chains of central nuclei in myofibers, increased NADH reactivity, degenerating fibers and AS alterations Neonatal lethality in mice expressing high levels of CELF1 Contribution of CELF1 overexpression to DM1 muscular phenotype [62]
TRECUGBP1 Insertion of Tet-responsive human CELF1 transgene; heart-specific rtTA expression Left ventricular systolic dysfunction and dilatation, AS alterations DM1-like phenotype limited to heart defects Contribution of CELF1 overexpression to DM1 heart phenotype [63]
TRECUGBP1 Insertion of Tet-responsive human CELF1 transgene; skeletal-muscle-specific rtTA expression Myofibers containing central nuclei, decreased muscle weight, impaired muscle function, AS alterations DM1-like phenotype limited to skeletal-muscle defects Contribution of CELF1 overexpression to DM1 skeletal-muscle phenotype [65]
TRECUGBP2 Insertion of Tet-responsive human CELF2 transgene; heart-specific rtTA expression No observed heart pathology; AS alterations similar to those observed in TRECUBP1 mice Mild heart phenotype Contribution of CELF2 overexpression to DM1 heart phenotype [64]
  (B) Transgenic Models with Repeat Expansion
  Mouse Model Generation Strategy (CTG)n DM1-Like Features Limitations Research Application Ref
  DM200 Insertion of a Tet-responsive expanded DMPK transgene where DMPK coding region is replaced by GFP 200 Ribonuclear foci; MBNL1 sequestration; AS alterations; myotonia, progressive cardiac conduction abnormalities Splicing alterations in the heart have not been described Study of DM1 phenotype associated with toxic CUG repeats; modeling muscle regeneration; test of therapeutic strategies [68][69][70]
  DM300 Insertion of a 45Kb human genomic fragment containing DMWD, DMPK and SIX5 genes from a DM1 patient ~300 Ribonuclear foci (skeletal muscle, heart and brain); myotonia; muscle atrophy; morphological abnormalities; changes in the distribution of MAPT/Tau protein isoform; defect in glucose metabolism High mortality; mild splicing alterations; intergenerational instability of CTG-repeat numbers Evaluation of DMPK transcript toxicity in different tissues [71][72]
  DMSXL Insertion of a 45Kb human genomic fragment containing DMWD, DMPK and SIX5 genes from a DM1 patient >1000 Ribonuclear foci; MBNL1 sequestration; AS alterations; deficits in motor performance; behavioral abnormalities; synaptic dysfunction; inhibition of exploratory activity and cerebellar glial dysfunction High mortality; severe body-weight reduction; interindividual variability; decreased transgene expression with aging; mild muscular phenotype Evaluation of DMPK transcript toxicity in different tissues and in multiple brain cell types; test of therapeutic strategies [23][73][74]
  HSALR Insertion of the human skeletal actin (HSA) gene including CTG repeats in the 3’ UTR ~250 Ribonuclear foci; AS alterations;
myotonia and muscle histopathology abnormalities (increase in central nuclei and variability in fiber size) after six months of age		 Limited to skeletal muscle; does not contain DMPK gene sequence; absence of muscle weakness Investigation of expanded-CUG-repeat toxicity
in muscle fibers; test of therapeutic strategies		 [75][76]
  LC15 Insertion of CTG expanded DMPK 3’ UTR downstream Luciferase gene driven by CMV-βA promoter 250–400 Ribonuclear foci, AS alteration and MBNL2 upregulation in the heart; reduced Na+ and K+ channel activity; ventricular arrhythmias DM1-like phenotype limited to heart defects Evaluation of biophysical mechanisms reproducing DM1-like electrocardiograph abnormalities [77]
  EpA960/
𝛼-MHC-Cre		 Insertion of CTG expanded DMPK exon 15 transgene containing Cre-responsive loxP sequences; heart-specific myosin Cre expression 960
(CTCGA-interrupted)		 Ribonuclear foci; MBNL1 sequestration; CELF1 protein upregulation; AS alterations;
cardiomyopathy, arrhythmias; systolic and diastolic dysfunction		 Does not reproduce CTG-repeat continuity; mouse model no longer available Evaluation of DMPK transcript toxicity and CELF1 overexpression in heart tissue [78]
  EpA960/
HSA-Cre		 Insertion of CTG expanded DMPK exon 15 transgene containing Cre-responsive loxP sequence; skeletal-muscle-specific Cre expression 960
(CTCGA-interrupted)		 Ribonuclear foci; MBNL1 sequestration; CELF1 protein upregulation; AS defects; myotonia and progressive muscle wasting, deficits in muscle performance and histopathological abnormalities Does not reproduce CTG-repeat continuity; mouse model no longer available Evaluation of DMPK transcript toxicity and CELF1 overexpression in skeletal tissue [79]
  EpA960/
CamKII-Cre		 Insertion of CTG expanded DMPK exon 15 transgene containing Cre-responsive loxP sequence; brain-specific Cre expression 960
(CTCGA-interrupted)		 Ribonuclear foci; MBNL1 sequestration; AS alterations; learning disability; neurotransmission dysfunction; brain atrophy and aging Does not reproduce CTG-repeat continuity; mouse model no longer available Identify mechanisms involved in CTG-dependent neuronal degeneration [80]
  TREDT960I/𝛼-MHC-rtTA Insertion of Tet-responsive expanded DMPK exons 11–15 transgene; heart-specific rtTA expression 960
(CTCGA-interrupted)		 Ribonuclear foci; MBNL1 sequestration; CELF1 protein upregulation; AS alterations ; arrhythmias Does not reproduce CTG-repeat continuity Study of alteration of ion transport and action potential in cardiomyocytes expressing toxic CUG [81][82]
  TREDT960I/
MDAF-rtTA		 Insertion of Tet-responsive expanded DMPK exons 11–15 transgene; skeletal-muscle-specific rtTA expression 960
(CTCGA-interrupted)		 Ribonuclear foci; MBNL1 sequestration; CELF1 protein upregulation; AS alterations; muscle wasting and myopathy Does not reproduce CTG-repeat continuity Study the mechanisms of CUG-repeat-induced muscle tissue loss [83]
Abbreviations: AS = alternative splicing; ChP = brain choroid plexus; CMVβA = cytomegalovirus enhancer/β-actin; GFP = green fluorescent protein; KO = knockout; MDAF = expression vector carrying regulatory sequences for the rat myosin light chain 1/3 gene; MHC = myosin heavy chain; Myo = myogenin; NADH = nicotinamide adenine dinucleotide; polyA = polyadenylation; rtTA = reverse tet transactivator.
Based on the assumption that the expanded DMPK transcript is the main cause of DM1 disease, many different mouse models expressing expanded CUG transcripts either ubiquitously or in specific tissues were generated to model the disease mechanisms (Table 1B). The multisystemic impact of CUG expansions is well recapitulated in DM200, DM300 and in DMSXL transgenic mice carrying the 3′ UTR portion or the entire human DMPK gene with CTG repeats of different lengths, the phenotype being more severe in mice with larger expansion [23][68][71]. In DM300 and DMSXL mice, transgene expression resulted in the accumulation of ribonuclear foci in various tissues and in the development of muscle weakness, behavioral abnormalities, growth retardation and perinatal mortality [23][71][72][73][74]. Recently, a mouse model constitutively expressing CTG repeats within the DMPK context was generated, which exhibited particularly high CUG expression in the heart (LC15). These mice reproduced DM1-like cardiac defects [77]. Skeletal-muscle-specific, heart-specific and brain-specific DM1-like features have been reproduced in mouse models expressing the repeat expansion in the respective tissues, either constitutively (HSALR) [76] or inducibly (EpA960 and TREDT960I mice strains) [75][78][79][80][81][82][83]. The tissue-specific phenotypes are usually strong and suitable for testing therapeutic molecules. However, at the same time, they do not recapitulate the multisystemic DM1 phenotype. In the inducible models, CTG repeats interrupted with stretches of CTCGA have been inserted in a portion of the DMPK human transgene. Interrupted repeats have the advantage of being more stable than CTG repeats, but may not exactly reproduce the human DM1 disease condition. Each of these mouse models exhibits advantages and limitations mostly depending on the temporal and spatial control of the transgene expression (detailed in Table 1). Taken together, transgenic mouse models have been crucial to understanding DM1 pathogenetic mechanisms and to testing therapeutic approaches.

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Germana Falcone
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Claudia Provenzano
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Fabio Martelli
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Beatrice Cardinali
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