Therapeutic Gene Modulation: Targeting Muscleblind’s Regulation by miRs

Published on Tue, 07/17/2018

Free Muscleblind (MBNL) protein levels are thought to be the linchpin in the pathogenesis of myotonic dystrophy. Sequestration of MBNL by binding to hairpin structures formed expanded DMPK RNA repeats leads to missplicing of mRNAs essential to the normal structure and function of the various tissues impacted by DM. Thus preservation of functionally significant intracellular levels of MBNL represents a viable strategy for therapy development. A new publication by Beatriz Llamusi, Ruben Artero, and colleagues (University of Valencia, Incliva Health Research Institute, and Joint Unit Incliva-CIPF) identifies mIR regulators of MBNL as potential therapy development targets for DM1 (Cerro-Herreros et al., 2018).

Identifying miR Suppressors of Mammalian MBNL

Multiple lines of evidence have established MBNL up-regulation as a potentially effective target for novel therapies in DM1. A prior study in a DM1 Drosophila model (Cerro-Herreros et al., 2016) showed reversal of missplicing events, reduced skeletal muscle pathology, improved muscle function, and increased lifespan via suppression of two micro RNAs (miRs) known to negatively regulate MBNL. A new publication from this same team extends these findings to mammalian cell and animal model systems.

Based on their prior work in the fly, the research team overexpressed a panel of miRs in HeLa cells, identifying those that reduced MBNL1 or MBNL2 transcript levels by a threshold of 4-fold or higher. miRs were selected for further analysis based on the HeLa assay data and in silico predictions of MBNL binding strength. Additional studies confirmed a miR subset (miR-96, miR-218, and miR-23b) as binding to the MBNL 3’-UTR and negatively impacting both MBNL transcript and protein levels.

Antagonizing miR-23b and miR-218 in vitro Stabilizes MBNL mRNA

Two of the miRs found to bind and suppress MBNL, miR-23b and miR-218, were also highly expressed in DM1myoblasts and muscle biopsies. Targeting DM1 myoblasts with antisense oligonucleotide (“antagomiR”) to miR-218 showed substantial (50% > controls) and dose-dependent increases in MBNL transcripts. By contrast, miR-23b anatagomiR did not show classic dose-response behavior, but was effective at a lower dose and exhibited clear mechanism of action in directly reducing miR-23b levels and thereby increasing MBNL transcripts. Preliminary toxicology studies suggested that the two antagomiRs were effective at concentrations below toxicity threshold.

Antagonizing miR-23b and miR-218 Rescues Molecular, Structural, and Functional Consequences of DM1

In a series of studies, the research team developed evidence to support antagonism of miR-23b and miR-218 as potentially viable paths toward a therapeutic for DM1. This evidence included: both antagomiRs were shown to rescue splicing defects in DM1 patient-derived cells; both restored normal cellular localization of MBNL protein; both increased Mbnl expression in HSALR mouse skeletal muscle; both rescued multiple splicing events in HSALR mice; and both improved skeletal muscle histopathology and reduced myotonia in HSALR mice. In additional studies, the research team showed continued miR target reduction and molecular, structural (skeletal muscle central nuclei), and functional (forelimb grip and myotonia) efficacy at 6 weeks after antagomiR injection.


This publication makes a compelling case that targeting the suppressive activity of miR-23b and miR-218 to up-regulate MBNL is a potential therapeutic strategy for DM1. Both miRs were found to be expressed in organ systems impacted by DM1 (skeletal muscle, heart, and CNS were assessed) and suppression of either modulated the molecular, structural, and/or functional consequences of DM1 in appropriate cell or animal models. There are several considerations before this target validation work can translate into the clinic, including, but not limited to, performance of a definitive proof of concept study (independent replication under rigorous conditions is essential for any therapeutic development effort) and either optimization of the currently limited intracellular delivery capacity of antisense oligonucleotides or development of small molecule inhibitors of miR-23b or miR-218.


Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila.
Cerro-Herreros E, Fernandez-Costa JM, Sabater-Arcis M, Llamusi B, Artero R.
Sci Rep. 2016 Nov 2;6:36230. doi: 10.1038/srep36230.

miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.
Cerro-Herreros E, Sabater-Arcis M, Fernandez-Costa JM, Moreno N, Perez-Alonso M, Llamusi B, Artero R.
Nat Commun. 2018 Jun 26;9(1):2482. doi: 10.1038/s41467-018-04892-4.

Senators Play Key Role in Getting DM Access to New Federal Research Funds

Published on Tue, 06/12/2018

MDF honored both Senator Dick Durbin and Senator Diane Feinstein with MDF Congressional Leadership Awards for their outstanding work and support in getting myotonic dystrophy added as an eligible research area in the U.S. Department of Defense (DOD) Peer-Reviewed Medical Research Program (PRMRP) for Fiscal Year 2018 (FY18). Senator Durbin serves as the Ranking Member on the Defense Subcommittee on Appropriations, which has oversight over Defense Health Programs like the PRMRP, and Senator Feinstein is a senior member of the subcommittee.

On a recent visit to Washington, DC, MDF community members Gareth and Jane Williams of Lake Forest, IL, along with MDF Board Chair Dr. Woodie Kessel, presented Senator Durbin with the MDF Congressional Leadership Award and enjoyed an informative and affable meeting discussing the impact that DM has had on the Williams family, Senator Durbin’s recent interest in gene editing and CRISPR, and their mutual affection for British television shows. The Senator and his staff reaffirmed their commitment to helping the DM community, and kept that promise a week later when he mentioned myotonic dystrophy to National Institutes of Health (NIH) Director Frances Collins at a Senate Appropriations hearing.

Dr. Kessel and MDF Board member and California resident Martha Brown were in Washington several weeks later to present Senator Feinstein with an MDF Congressional Leadership Award. Senator Feinstein and her staff, in particular Chris Gaspar have been incredibly helpful to MDF as we navigate through the FY18 PRMRP process and as we continue our efforts to ensure that myotonic dystrophy remains included in the PRMRP in Fiscal Year 2019. Woodie and Martha thanked Chris for his continued engagement on this issue, and the group had a productive meeting where they discussed PRMRP strategies going forward.

MDF remains deeply grateful to both Senators and their staff, and we look forward to continuing to work together in the future to advance DM research.

Get Involved!

For more information on MDF's advocacy program and how you can get involved, watch a video on our recent advocacy update and training.

Questions? Contact MDF.

Thank Senators Durbin and Feinstein for their Work

Senator Durbin

Send his office an email.
Call his office at 202-224-7703.

Senator Feinstein

Send her office an email.
Call her office at (202) 224-3841.


Mechanisms of Muscle Wasting in DM1

Published on Tue, 06/12/2018

Many Muscular Dystrophies, Yet Diverse Paths to Muscle Atrophy

By definition, the muscular dystrophies are diseases that cause progressive muscle atrophy and weakness. Often, as in myotonic dystrophy, other body systems are involved as well, but it is the atrophy and weakness that are the traditional hallmarks of the disease. Loss of skeletal muscle mass has been linked to variety of causes across the various types of muscular dystrophy—from the breakdown of the sarcomere and calcium-triggered proteolysis in mutations of the dystrophin-dystroglycan complex (e.g., dystrophinopathies and some forms of LGMD) to mutations in nuclear lamins, which disrupt intracellular signaling (e.g., EDMD) to mutations that alter sarcolemmal repair (e.g., dysferlinopathies). By contrast, the molecular mechanisms linking expanded repeat tracts to muscle atrophy in DM are largely unresolved.

Insights from a New Mouse Model

Dr. Ginny Morriss, an MDF fellow, and her mentor, Dr. Tom Cooper, and their colleagues at Baylor College of Medicine have developed a novel mouse model and used it to gain insights into the molecular mechanisms behind skeletal muscle wasting in DM1. Their findings suggest that disruption of specific cell signaling pathways may be an important contributor to muscle atrophy in the mouse model.

The Baylor research team developed a skeletal muscle-specific, tet-inducible mouse expressing 960 CUG repeats in the context of human DMPK exons 11-12 (CUG960 mouse). While the mice showed substantial skeletal muscle atrophy—reduced muscle weight and histologic abnormalities—and MBNL-containing nuclear foci, the splicopathy that normally characterizes DM1 was mild. Likewise, Celf1,GSK3β, or cyclin D3 levels did not show significant changes in induced CUG960 mice.

Induction of CUG960 induction in utero or at postnatal day 1 produced similar skeletal muscle outcomes. Muscle loss was also observed when CUG960 mice were induced at 6 weeks of age, but effects were less consistent across muscle groups. Additional assays failed to detect alterations in total protein synthesis levels as an underlying mechanism. Turning off CUG960 expression ten weeks after induction at postnatal day 1 reversed the skeletal muscle effects in some, but not the most severely affected, muscle groups.

DM1 Spliceopathy Does Not Fully Explain Muscle Wasting in DM1

Since the splicing defects in induced CUG960 mice were mild, the research team sought to understand other potential mechanisms for skeletal muscle atrophy. Reverse phase protein array analysis, an antibody-based assay to quantitatively assess large numbers of biologic samples for alterations in signaling pathways, identified substantial alterations in protein abundance—77 proteins up-regulated and 2 down-regulated. The protein assay findings correlated with other measures of muscle wasting on a sample-by-sample basis. Altered phosphorylation of some signaling pathway components was also noted.

Observed disruptions in signaling pathway components were interpreted as reflective of a deregulation of PDGFRβ receptor signaling and the PI3K/AKT pathways, along with prolonged activation of AMPKα signaling. The research team obtained similar results in analyses of DM1 patient muscle biopsy material, providing additional validation for the mouse model studies.

Overall, the alterations in cellular signaling support the concept that alterations in the balance of anabolic and catabolic pathways that normally maintain muscle mass, independent of MBNL-triggered splicing abnormalities, is an important contributor to muscle atrophy in DM1. Modeling of skeletal muscle atrophy in DM then must take into account mechanisms dependent and independent of reductions in free MBNL levels.


Mechanisms of skeletal muscle wasting in a mouse model for myotonic dystrophy type 1.
Morriss GR, Rajapakshe K, Huang S, Coarfa C, Cooper TA.
Hum Mol Genet. 2018 May 16. doi: 10.1093/hmg/ddy192. [Epub ahead of print]

Similar Molecular Mechanisms, But Divergent Phenotypes in DM1 and DM2—Why?

Published on Tue, 06/12/2018

Although the nature and locations of the repeat expansions are different for DM1 and DM2, the underlying molecular mechanism, sequestration of MBNL and consequent missplicing of a wide range of transcripts, is ostensibly the same. It then begs the question, why are the phenotypes of DM1 and DM2 different?

Understanding Molecular Mechanisms in DM1 and DM2

One “logical” interpretation for the milder phenotype in DM2 is that CCUGexp repeats may be less toxic. Perhaps MBNL is not sequestered as efficiently by DM2 repeats (hence DM2 patients typically have much longer repeats) and thus not depleted to the same extent as in DM1. But there are many potential alternative hypotheses, including differences due to the genomic locations of the expanded repeats or the presence of as yet unknown modifiers that act upon the expanded repeat transcript or other potential targets upstream of the ensuing splicing anomalies.

Drs. Beatriz Llamusi (University of Valencia) and Nicolas Charlet-Berguerand (Université de Strasbourg) and colleagues have identified an RNA binding protein that acts to reduce MBNL depletion in DM2, but not in DM1. Their data suggests a molecular explanation that, at least in part, reconciles the phenotypic differences between DM1 and DM2.

rbFOX1 binds CCUGexp, but not CUGexp RNA

To assess whether alternative RNA binding proteins may be more efficient at binding to CCUGexp versus CUGexp, and thereby mitigate MBNL sequestration in DM2, the research team conducted an unbiased assay for C2C12 cell nuclear extract interactions with the two expanded repeat sequences. They identified several proteins that bound to the expaned repeat sequences, but, among the hits, only rbFOX family RNA binding proteins interacted with CCUGexp, but not CUGexp. They further showed that rbFOX1 localizes to nuclear foci only in cells containing the DM2 repeats.

The research team them utilized gel-shift and UV-crosslinking experiments to demonstrate the mechanism by which rbFOX1 is retained in nuclear foci containing CCUGexp sequences—these studies confirmed a direct interaction between rbFOX1 and CCUGexp.

rbFOX1 is Bound to CCUGexp in DM2 Patient Cells

In additional experiments, the research team established that rbFOX1 co-localized with CCUGexp–containing foci in primary muscle cultures derived from DM2 patients, as well as in sections from DM2 patient skeletal muscle. Yet, additional studies showed that rbFOX1 was not immobilized in CCUGexp RNA foci in the same manner as MBNL. rbFOX1 was not detected in similar nuclear analysis of DM1 patient cells or tissues.

Excess rbFOX1 Displaces MBNL from CCUGexp and its Overexpression Corrects Splicing and Muscle Atrophy

Although rbFOX1 and MBNL both bind CCUGexp, their interactions may or may not be competitive. But, expression of an excess of either rbFOX1 or MBNL was shown to compete the other away from binding to the DM2 expanded repeat; depletion of rbFOX1 increased the localization of MBNL to CCUGexp. These data are consistent with the hypothesis that rbFOX1 and MBNL compete for the same binding site.

The next step was to test whether an overabundance of rbFOX1 could mitigate the downstream effects of MBNL depletion. Overexpression of rbFOX1 in C2C12 cells carrying CCUGexp resulted in partial correction of splicing of Clcn1 and Tnnt2 mini-genes, while reduction of rbFOX1 by siRNA increased the splicing defect. Finally, the research team overexpressed rbFOX1 in Drosophila models of DM1 and DM2 and showed rescue of skeletal muscle atrophy only in the DM2 model.

Taken together, the investigators here provide a molecular model to explain, at least in part, the phenotypic differences between DM1 and DM2. Reduced sequestration of MBNL by CCUGexp versus CUGexp repeats by rbFOX1 would mitigate the splicing defects and thereby potentially contribute towards the milder pathology in DM2.


rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophytype 1/type 2 differences.
Sellier C, Cerro-Herreros E, Blatter M, Freyermuth F, Gaucherot A, Ruffenach F, Sarkar P, Puymirat J, Udd B, Day JW, Meola G, Bassez G, Fujimura H, Takahashi MP, Schoser B, Furling D, Artero R, Allain FHT, Llamusi B, Charlet-Berguerand N.
Nat Commun. 2018 May 22;9(1):2009. doi: 10.1038/s41467-018-04370-x.

Important New Review Articles on DM

Published on Tue, 06/12/2018

Frontiers of Neurology Publishes a Series of DM Reviews

A new set of six review articles is being published on various aspects of DM. These articles comprise a special issue of Frontiers in Neurology, entitled: ‘Beyond Borders: Myotonic Dystrophies—A European Perception', co-edited by Profs. Giovanni Meola and Benedikt Schoser. Together, these articles represent an important new reference source for DM stake-holders.

Coverage for A Broad Range of DM Topics

Wenninger et al. (2018) note the difficulties caused in interpretation of the highly variable and multi-systemic phenotypes exhibited in DM1 and DM2. They have focused this review on what they define as the ‘core clinical features’ of DM, with the intent that this perspective can facilitate early diagnosis and treatment.

André et al. (2018) review the evidence that DM1 and DM2 have an impact throughout the skeletal muscle life cycle, influencing its development, growth, regenerative capacity, and premature aging. They suggest that invoking the well-recognized involvement of the expanded repeat and RNA dysfunction alone may be too simplistic an explanation for the pathogenesis of skeletal muscle in DM.

Matloka et al. (2018) take on the important issue of using cell-based systems to understand pathogenic mechanisms and to screen candidate therapeutics for DM. Their article reviews the history of utilization of cellular models in DM1 and catalogs models that are available for basic science and translational research.

López-Morató et al. (2018) recognize the extensive efforts to therapeutically target the depletion of MBNL by expanded repeat DMPK transcripts using large molecules specifically designed for the target. In their review, they look beyond those efforts at other targets that have been engaged by small molecule drug discovery and development programs for DM1.

Two other articles remain to be published—see below.

Publication Status

The complete articles by Chakraborty et al. (2018) and Mahyera et al. (2018) are not yet available, although the abstracts are on the Frontiers of Neurology website. The work by Dr. Chakraborty and colleagues discusses use of fly models to understand the molecular and physiologic pathogenesis of the heart in DM1 and DM2, and the small molecules that have been evaluated for cardiac dysfunction in these models. The work by Dr. Mahyera and colleagues discusses the population distribution and structure of expanded repeat tracts in DM2 patients in Germany.

Four of the article pdfs are available, open-access, at the Frontiers in Neurology website. The other two articles will be posted to this site when published.


Abnormalities in Skeletal Muscle Myogenesis, Growth, and Regeneration in Myotonic Dystrophy
André LM, Ausems CRM, Wansink DG, Wieringa B.
Front Neurol. 2018 May 28;9:368. doi: 10.3389/fneur.2018.00368. eCollection 2018. Review.
[This article is available as a pdf on the journal website but was not yet listed in PubMed at the time of this writing, so the link to the PubMed abstract was not yet available.]

Modelling of myotonic dystrophy cardiac phenotypes in Drosophila.
Chakraborty M, Llamusi B, Artero R.
Front Neurol. doi: 10.3389/fneur.2018.00473. eCollection 2018. Review.
[This article was not listed in PubMed, and it is listed as ‘provisionally accepted’ on the Frontiers in Neurology website. Thus, the citation is incomplete and neither the PubMed link nor the pdf were available at the time of this writing.]

Small Molecules Which Improve Pathogenesis of Myotonic Dystrophy Type 1.
López-Morató M, Brook JD, Wojciechowska M.
Front Neurol. 2018 May 18;9:349. doi: 10.3389/fneur.2018.00349. eCollection 2018. Review.

Distribution and Structure of DM2 repeat tract alleles in the German Population.
Mahyera AS, Schneider T, Halliger-Keller B, Schrooten K, Hörner E-M, Rost S, Kress W.
Front Neurol. doi: 10.3389/fneur.2018.00473. eCollection 2018. Review.
[This article was not listed in PubMed, and it is listed as ‘provisionally accepted’ on the Frontiers in Neurology website. Thus, the citation is incomplete and neither the PubMed link nor the pdf were available at the time of this writing.]

Cells of Matter-In Vitro Models for Myotonic Dystrophy.
Matloka M, Klein AF, Rau F, Furling D.
Front Neurol. 2018 May 23;9:361. doi: 10.3389/fneur.2018.00361. eCollection 2018. Review.

Core Clinical Phenotypes in Myotonic Dystrophies.
Wenninger S, Montagnese F, Schoser B.
Front Neurol. 2018 May 2;9:303. doi: 10.3389/fneur.2018.00303. eCollection 2018. Review.

Microsatellite Expansions Selectively Trigger Intron Retention

Published on Tue, 05/08/2018

Microsatellite expansions triggering a variety of neurological disorders occur in both coding and non-coding regions. Trinucleotide CNG expansions (e.g., DM1) predominate in exonic and UTR regions, while intronic expansions can vary from tri- to hexanucleotide repeats and are associated with eight diseases that include DM2, C9orf72 ALS/FTD, and Fuchs endothelial corneal dystrophy. How the sequence and differential localization of microsatellite expansions impacts disease mechanisms is not fully known.

Dr. Maury Swanson (University of Florida), Łukasz Sznjader, a 2016-2017 MDF Fellow, and researchers from the University of Rochester, Houston Methodist Hospital, and Adam Mickiewicz University in Poland, have recently evaluated the concept that intronic microsatellite expansions themselves act as a trigger to host gene intron retention and thereby to the pathogenesis of disease. In this study, they assessed a variety of neurological diseases linked to GC- and A/AT-rich intronic expansions.

The presence of microsatellites in introns alone conveys very low risk of expansion and triggering of inherited diseases—of 80,000 intronic microsatellites with expansion potential, only 0.01% are actually known to expand and cause disease. The research team mapped the intronic localization of both pathogenic expanded microsatellites and unexpanded repeats and demonstrated a bias of disease-causing microsatellites toward splice sites and thereby represent potential triggers for splicing alterations and intron inclusion. CNBP intron 1 inclusion as determined from a DM muscle RNA-seq database and patient biopsy samples was substantially elevated in contrast to intron inclusion across a broad range of neuromuscular disease controls. Notably, only CNBP intron 1 (the site of the microsatellite expansion) was spliced in, while other downstream CNBP introns were not. Further studies showed that intron 1 retention was not a developmentally regulated event.

Dr. Swanson and colleagues then tested the hypothesis that CNBP intron 1 inclusion in DM2 patient peripheral blood could be used as a disease biomarker. These studies confirmed intron inclusion in DM2, but not in controls, in a repeat length-dependent fashion. At this point it was as yet unclear whether the expanded CCUG repeat itself was mechanistic in altering CNBP splicing to include intron 1. A mouse reporter gene model was used to confirm that intron inclusion was driven by CCUGexp in a repeat length-dependent manner. Finally, the research team showed that intronic retention occurred only for GC- (e.g., DM2), but not A/AT-rich (e.g., Friedrich’s ataxia), microsatellite expansion diseases.

Taken together, this report identifies the molecular mechanisms underlying intronic retention in DM2 and emphasizes its value as a putative biomarker, particularly since intronic retention is so easily assessed in peripheral blood using routine RT-PCR assays. Such an approach to disease detection is much more efficient than conventional genetic strategies as a means of mapping heritable microsatellite expansion disorders and can be broadly used in discovery of the genetic basis of currently undiagnosed diseases.

Intron retention induced by microsatellite expansions as a disease biomarker.
Sznajder ŁJ, Thomas JD, Carrell EM, Reid T, McFarland KN, Cleary JD, Oliveira R, Nutter CA, Bhatt K, Sobczak K, Ashizawa T, Thornton CA, Ranum LPW, Swanson MS.
Proc Natl Acad Sci U S A. 2018 Apr 2. pii: 201716617. doi: 10.1073/pnas.1716617115. [Epub ahead of print]

Quantifying Mutant mRNA in DM1 and DM2

Published on Tue, 05/08/2018

Robust, reliable, and reproducible endpoints are essential for decision making in early stage drug discovery and development programs. Given the high level of understanding of molecular events in the pathogenesis of DM, assays that focus on careful quantification of disease-causing molecules are essential for go/no-go decision making in drug screening programs. Thus far, methodology for reliable quantification of mutant RNA copy number, although a critically important tool for drug developers, has been a question mark. A recent study utilized a battery of techniques (Northern blotting, reverse transcriptase-quantitative polymerase chain reaction, RNA-sequencing and fluorescent in situ hybridization analyses) to demonstrate a very low abundance—one to a few dozen molecules per cell—Dmpk/DMPK mRNA in mouse model and patient samples (Gudde et al., 2016).

Dr. David Brook (University of Nottingham) and colleagues have reported out on studies of methodology to precisely quantify (a) the absolute numbers of mutant RNA transcripts and (b) alternative splicing in DM1 and DM1 patient cell samples (Wojciechowska et al., 2018). In these studies, they evaluated the potential value of medium throughput technologies—Multiplex Ligation-Dependent Probe Amplification (MLPA) and droplet digital PCR (ddPCR)—that could be feasibly applied in drug development programs.

To quantitatively assess alternative splicing, the research team designed assay methodology that would evaluate eight key splicing events in DM1 and DM2 patient skeletal muscle biopsy samples. MLPA used two probes for each exon (exon-On and exon-Off). Likewise, ddPCR utilized dual TaqMan probes for each splicing event, specific to exon-On and exon-Off. Transcripts evaluated met specific selection criteria and represented known changes at different disease stages. Both MLPA and ddPCR met rigorous criteria for splicing quantification (sensitivity, reliability) and results showed strong correlation across the two methods.

To achieve absolution quantification of DMPK mRNA copy number, the group optimized ddPRC assays and used them to characterize DM1 skeletal muscle biopsies and cultured fibroblasts. Mutant and wild type DMPK transcripts were differentiated on the basis of the rs527221 G>C SNP in exon 10. Results suggested that highly sensitive, reproducible, and accurate quantification was achievable even with the low mutant mRNA copy number (15-20 molecules/cell) that characterize DM.

In addition to assessing the appropriateness of MLPA and ddPCR as biomarker assays for myotonic dystrophy, the research team demonstrated that the absolute abundance of mutant DMPK transcripts in the nuclear foci of DM1 patient fibroblasts was very low. Thus, sensitive and reliable assays that can avoid floor effects are important for the field.

Finally in situ fluorescence hybridization was used to estimate mutant DMPK mRNA in individual nuclear foci. The median number of mutant CUG transcripts per nuclear foci was between two and four, with between four and six total foci detected per DM1 patient fibroblast.

Taken together, both MLPA and ddPCR technologieswere shown to produce highly reproducible and simultaneous assessment of genes aberrantly spliced in DM, and ddPCR was an effective measure of absolute mutant DMPK transcript copy numbers per cell. Validation of this methodology addresses an important gap in tools available for the therapy development pipeline for DM.


A low absolute number of expanded transcripts is involved in myotonic dystrophy type 1 manifestation in muscle.
Gudde AE, González-Barriga A, van den Broek WJ, Wieringa B, Wansink DG.
Hum Mol Genet. 2016 Apr 15;25(8):1648-62. doi: 10.1093/hmg/ddw042. Epub 2016 Feb 16.

Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy.
Wojciechowska M, Sobczak K, Kozlowski P, Sedehizadeh S, Wojtkowiak-Szlachcic A, Czubak K, Markus R, Lusakowska A, Kaminska A, Brook JD.
Sci Rep. 2018 Apr 12;8(1):5885. doi: 10.1038/s41598-018-24156-x.

Genome Editing: The Hope and the Hype

Published on Thu, 05/03/2018

Some MDF community members may have watched the news show, 60 Minutes, on April 29th. The show featured a segment titled: "CRISPR: The Gene-Editing Tool Revolutionizing Biomedical Research" (if you missed it, the transcript is available here). 60 Minutes took a thoughtful approach in choosing the words " tool" and "research" for its segment title. Understanding those two words is the key to parsing hope from the hype in genome-editing technology.

Evaluating Genome-Editing Technology

MDF received a generous donation in late 2017 to help evaluate whether genome-editing technologies, like CRISPR, have the potential to translate into a safe and effective therapy for DM1. Both the donors and MDF are on the same page in understanding what Dr. Eric Lander (President and Founding Director, Broad Institute of MIT and Harvard) said so clearly about CRISPR on 60 Minutes: “I [want to] always balance hope versus hype here. While it's not [going to] affect somebody who might be dying of a disease today, this is [going to] have a real effect over the course of the next decade and couple of decades. And for the next generation, I think it'll be transformative.”

Genome editing includes a range of technologies—CRISPR is just one of several with the potential to address inherited human diseases. For this technology to achieve its promise for patients, genome-editing reagents have to be delivered to the correct body tissues and, once there, edit the affected genes in efficient and safe ways. All three parts of that equation—delivery, efficient editing, and safety in avoiding unintended damage to other genes—must be optimized. It’s important to remember that CRISPR is a natural defense mechanism used by bacteria to kill invading viruses. Drug developers need to ensure that a technology designed to kill must be transformed into one that very selectively edits defective human genes in a highly controlled manner.

Strategies for Research

Because genome-editing technology must continue to evolve before this research tool is transformed into effective therapies, MDF organized an expert workshop to understand the current state of the science and identify opportunities and barriers to moving forward. On April 17, MDF convened a day-long panel of 14 experts from universities, companies and Federal agencies (NIH and FDA), along with MDF staff and the donors, as a first step toward understanding how to foster research that evaluates and optimizes genome-editing strategies to the specific needs of DM1.

Workshop participants discussed how to optimize genome-editing strategies, how to best deliver genome-editing reagents to the body tissues where they need to act, how to evaluate the efficacy and safety of genome-editing tools in patient cell and animal models, and, finally, how to best implement the evaluation and development of genome-editing in the context of the unique genetics of and patient needs in DM1. The small group atmosphere and tightly-focused discussions at the workshop led to a wealth of information for MDF to use in the design of a request for proposals (RFP) soliciting research grant applications, evaluation of those applications and guidance of the funded research project(s).

Moving Forward

CRISPR and the other genome-editing technologies have considerable potential as an effective therapeutic for the approximately 7,000 inherited diseases that are known today. The hope for DM1 lies in the potential to remove the expanded repeat from the DMPK gene and thereby mitigate or eliminate many of the disease symptoms. But potential is not a drug. It’s important to foster hope while knowing that any excessive hype is not realistic at the current stage of research. MDF is launching this research program from a very informed perspective and will continue to relay advances from the program to the patient, family and research communities.

Questions? Contact MDF at

Determining DM Patients at Greatest Risk for Cardiac Problems

Published on Thu, 05/03/2018

When cardiologist Dr. William J. Groh examined a young woman with an arrhythmia in 1995, he thought it was unusual. She did not have known heart disease or heart failure that would normally be associated with a serious heart rhythm disturbance. What she did have, though, was myotonic dystrophy (DM).

The case sent Dr. Groh searching through the available medical literature. At the time, it was not established that such heart complications were common among myotonic dystrophy patients. He found papers discussing the issue, but there were no large studies that sought to answer fundamental questions about cardiac involvement in myotonic dystrophy.

Dr. Groh, who was then a fellow at Oregon Health & Science University in Portland, OR, decided to undertake a large natural history study of myotonic dystrophy type 1 patients. The goal was to determine in the general myotonic dystrophy population what the risk of heart problems was, whether there were predictors of such problems that could identify patients at greater risk, and what could be done prevent some of the adverse outcomes.

When the 11th meeting of the International Myotonic Dystrophy Consortium (IDMC-11) convened in San Francisco September 5–9, 2017, Dr. Groh was one of the researchers who presented.

“It was an excellent meeting,” Dr. Groh said. “It brought together a world-class group of leaders in the research and care of myotonic dystrophy. Every disease should have this type of support. Researchers, clinicians, patients, and advocacy groups all get together in one meeting, so it’s very important.”

Among the issues of greatest concerns for Dr. Groh are the need for additional research to identify the best strategies for addressing myotonic dystrophy type 1 (DM1) patients at risk for sudden death from arrhythmias, and the need for employing interdisciplinary teams to address the complexities of health issues DM patients face.

Study Launched

In April 1997, after moving to Indiana University, Dr. Groh began work on a major natural history study of DM1 patients. At that time, he believed that efforts to determine how to identify DM1 patients who were at high risk for sudden death were stymied because the populations for previous studies were too small and not adequately representative of the broad DM1 population. As a result, he said it left unclear the best way to diagnose and treat DM1 patients at greatest risk of sudden death.

The study, which enrolled 406 patients at 23 neuromuscular disease clinics in the United States, was the first such study to use genetic testing to confirm that all of the participants had DM1. It showed that some adult patients with the disease were at high risk for sudden death. It also found that severe electrocardiographic (EKG) abnormality and a clinical diagnosis of a sustained atrial arrhythmia were independent predictors of sudden death.

“We showed that we could use a simple EKG to help determine who was at high risk for heart problems in myotonic dystrophy,” he said. “Our work allowed us to take a general myotonic dystrophy population and screen people.”

He said if a younger person with myotonic dystrophy came in and had a normal electrocardiogram and no other heart findings on examination, the chances that they would have a problem relating to their heart in the next five years would be low. However, if they had significant abnormalities on their electrocardiogram, it was a cause for concern.

Differing Opinions

French researchers, he said, had been doing excellent work in parallel with his study. However, their work supported a greater use of prophylactic pacemakers in patients before serious heart problems are manifest. Dr. Groh’s work also showed the value of pacemakers but questioned whether the risk of sudden death was still excessive despite the presence of a pacemaker.

In fact, Dr. Groh has raised the issue of whether an implantable cardioverter-defibrillator (ICD) that can act as a pacemaker as well as treat rapid ventricular arrhythmias would be a better way to protect patients. In a paper published in June 2008, his work suggested that patients might benefit from an ICD.

Nevertheless, Dr. Groh cautions that his study was observational and that the French studies carefully examined a high-risk population that received pacemakers and showed benefit. He believes the question of whether the use of a pacemaker or ICD would allow patients to live longer and live a better life remains unresolved. “That’s another study that needs to be done,” he said.

“I think the next important trial in the United States would look at a high-risk population of myotonic dystrophy patients and treat them with an ICD and look at outcomes,” he said. “What we have found so far is that many U.S. doctors are putting pacemakers and ICDs in patients with myotonic dystrophy and we’d like to show, in fact, exactly which patients can benefit from this.”

A Team Approach

Dr. Groh’s interest in arrhythmias comes naturally. As an undergraduate student at Marquette University, he majored in electrical engineering. Today, as a cardiologist, a cardiac electrophysiologist, a professor at the Medical University of South Carolina, and the chief of medicine at the affiliated Veterans Affairs Medical Center, he focuses broadly on patients with heart rhythm disturbances, not just patients with DM.

He said that many cardiologists do not understand heart involvement in myotonic dystrophy. They may see just one or two myotonic patients and may not recognize the relevant issues. Dr. Groh said he’s always happy to get on his “soapbox” to preach about the need for a team of specialists to care for myotonic dystrophy patients.

“Because so many systems are affected by myotonic dystrophy, when you are approaching these individuals you have to deliver multidisciplinary care,” he said. “The team should include a neurologist, a physical therapist, a pulmonologist because of lung involvement, and a cardiologist. Over the last decade, we’ve seen more of these multidisciplinary clinics set up and this is important for delivery quality care to DM families.”

Using iPSC-Derived Cardiomyocytes to Understand DM1

Published on Tue, 04/10/2018

Induced pluripotential stem cell (iPSC) technology has provided opportunities to better understand disease mechanisms as well as to facilitate drug discovery and development programs. With the attendant molecular, cellular, and disease backgrounds of readily expandable, patient-derived cells, iPSC “disease-in-a-dish” models have supported the translation of candidate therapeutics from discovery into clinical testing.

As long as limitations are recognized, such as potential genetic and epigenetic divergence from primary cells, iPSC modeling of myotonic dystrophy (DM) may be essential to mechanistic understanding and development of treatments for the disease, particular when patient tissues are difficult to impossible to access (e.g., CNS and heart). MDF has facilitated the development of human DM1 iPSC lines, now available through RUCDR Infinite Biologics, with DM2 lines to follow.

Development of iPSC-Derived Cardiomyocytes

Reported in a newly published study, Dr. Federica Sangiuolo (University of Rome Tor Vergata) and colleagues have developed iPSC lines from two DM1 patients and two healthy controls. They characterized cardiomyocytes derived from these lines, with the goal of determining whether they could recapitulate at least some of the major traits of the DM1 heart. Myocyte characterization included RT-qPCR to quantify RNA expression, FISH and IF for nuclear foci, RT-PCR for splice variants, whole-cell patch clamp to characterize cardiomyocyte electrophysiology, and atomic force microscopy (AFM) to characterize biomechanical traits.

Traits of iPSC-Derived Cardiomyocytes—DM1 versus Wild Type

Most iPSC-derived cardiomyocytes exhibited a ventricular-like phenotype. DM1-derived cardiomyocytes contrasted with wild type controls in expressing nuclear foci and mis-slicing of each transcript evaluated (MBNL1, MBNL2, TNNT2 and SCN5A); DM1 derivatives also showed down-regulation of key cardiac ion channel transcripts (CACNA1C, KCNH2, KCNQ1, KCND3, and SCH5A) compared with controls. The research team noted novel differences in nuclear morphology in DM1 derivatives, potentially related to altered expression of lamin A.

Whole-cell patch clamp recordings of disaggregated single cardiomyocytes showed an abnormal electrophysiological profile only in the DM1 derivatives. DM1 ventricular myocyte-like derivatives exhibited lower spontaneous action potential rates, lower peak amplitude, and longer time to reach peak from threshold. Electrophysiologic parameters of DM1 cardiomyocytes could be altered by drugs targeting cardiac ion channels.

Using AFM, the research team identified differences in beat frequency and synchronicity of beats in DM1 iPSC-derived cardiomyocytes—describing the overall pattern as indicative of instability, with reduced frequency and the appearance of non-synchronous oscillation patterns when compared to wild type. The predominance of ventricular-like myocytes, and relative absence of nodal or other conduction system-related cardiomyocytes, may make it more difficult for iPSC-based models to be informative of the conduction system-specific abnormalities known to characterize DM1.

Overall, DM1 iPSC-derived cardiomyocytes recapitulated the main morphologic and molecular markers of the disease, including CTG expansions, nuclear foci, splicopathy of the mRNAs evaluated, and altered expression of several known cardiac ion channels. Alterations in electrophysiological parameters and biomechanical behavior were interpreted as consistent with unstable beating.


Modelling the pathogenesis of Myotonic Dystrophy type 1 cardiac phenotype through human iPSC-derived cardiomyocytes.
Spitalieri P, Talarico RV, Caioli S, Murdocca M, Serafino A, Girasole M, Dinarelli S, Longo G, Pucci S, Botta A, Novelli G, Zona C, Mango R, Sangiuolo F.
J Mol Cell Cardiol. 2018 Mar 15. pii: S0022-2828(18)30083-X. doi: 10.1016/j.yjmcc.2018.03.012. [Epub ahead of print]