Participants are often frustrated to find that they have limited to no access to their own data from a clinical study or interventional clinical trial. The operative phrase for the participant here is “their own data,” that results from the time, effort, and risks the patient undertakes to participate in clinical research. While their data may collectively contribute toward improving future clinical trials or provide the basis for regulatory decisions for a candidate therapeutic, the study participant views the data as having personal value.

The frustration for participants in learning little of study outcomes lies in feeling abandoned just as the study punch line becomes evident. Yet, potential patient confusion that any “research results” may automatically have reliable clinical value is a confounder to full transparency. To address this controversial issue, the NAS was asked by the Centers for Medicare and Medicaid Services (CMS), the Food and Drug Administration (FDA), and the National Institutes of Health (NIH) to develop a roadmap toward balancing the expectations of participants in clinical research with a careful risk-benefit assessment of the practice of releasing individual results to study participants.

NAS Report Advocates for Better Communication

The NAS put together a committee, bringing together experts in clinical research, informatics, stakeholder engagement, and biomedical ethics (patient representatives were included as reviewers of the report), that worked for a year to produce a 380-page report entitled: “Returning Individual Research Results to Participants: Guidance for a New Research Paradigm.” The report PDF is available at no charge on the NAS website (see link below).

Overall, the NAS report states: “The responsible return of individual research results requires careful forethought and preparation. Thus, the committee recommends that investigators include plans in study protocols that describe whether results will be returned and, if so, when and how and that research sponsors and funding agencies require that applications for funding consistently address the issue. Additionally, institutions and IRBs should develop policies to support the review of plans to return individual research results.” The decision about communicating research results to the patient was conceptualized by the committee as balancing the perceived value of the data to the participant with the feasibility of returning individual data in an understandable fashion. The demands that increased communication will place on the research enterprise are not inconsequential, but the potential benefits for clinical research through improved engagement and trust are considerable.

The NAS report highlights 12 specific recommendations to: facilitate evaluation of the risks and benefits of return individual research results to patients, establish biospecimen management procedures to ensure quality management, establish a decision making process for return of individual research results, ascertain patients preferences and values, guide planning for return of individual research results, effectively communicate individual research results to participants, and help reshape the regulatory environment.

Toward Better Clinical Studies

Taken together, the report seeks to implement changes that will improve collaboration and transparency between clinical investigators and research participants in order to improve the quality and effectiveness of clinical research. The benefits of improved engagement of stakeholders in the clinical research enterprise (e.g., patient-centered research) are already evident in improved study recruitment and retention, evolution of clinically meaningful study endpoints, and, ultimately, more effective care and novel therapeutics. With this report, NAS has broached the next logical step in committing to stakeholder communication. MDF encourages clinical researchers working on DM to read and carefully consider the NAS report.

References:

Returning Individual Research Results to Participants: Guidance for a New Research Paradigm: https://www.nap.edu/catalog/25094/returning-individual-research-results-to-participants-guidance-for-a-new

A series of additional NAS reports under the topic of “Exploring the Treatment of Research Participants” can be found at: https://notes.nap.edu/2018/07/12/treatment-research-participants/.

The Premise and the Plan

OPTIMISTIC (Observational Prolonged Trial In Myotonic dystrophy type 1 to Improve Quality of Life—Standards, a Target Identification Collaboration) [Link to archived site], an international, multi-center randomized clinical study funded by the European Commission and coordinated by Dr. Baziel van Engelen (Nijmegen, Netherlands), has achieved the milestone of publication of results in Lancet Neurology. A major driving factor for the study was the relative lack and small size of prior clinical trials in DM1. Thus, in addition to directly testing the value of exercise therapy and cognitive behavioral therapy against standard of care, the premise of OPTIMISTIC included gaining insights into outcome measures, use of genetic analysis and cardiac screening, and putative biomarkers to improve clinical trial readiness for DM1.

The specific goals of the OPTIMISTIC trial were to evaluate and compare: (a) exercise therapy to maintain or improve patient functional capacity and (b) cognitive behavioral therapy to simulate an active lifestyle and improve skeletal muscle function. Collectively, the interventions were tested as to their ability to improve DM1 patient quality of life. The intent of the OPTIMISTIC consortium is that study data may prove useful in providing clinical management guidance to both practitioners and patients to reduce the devastating symptom of fatigue and thereby improve quality of life.

The Trial and Outcomes

A total of 255 adult subjects with genetically confirmed DM1 and severe fatigue were recruited into OPTIMISTIC at four neuromuscular centers and randomly assigned to cognitive behavioral therapy plus standard of care or standard of care alone; a subset of the cognitive behavioral therapy plus standard of care group also received a graded exercise module. The primary outcome measure was 10-month change from baseline in the DM1-Activ-c scale.

The cognitive behavioral therapy group showed an adjusted mean difference improvement of 1.53 points in DM1-Activ-c versus an adjusted mean difference decline of 2.02 in the standard of care alone group. Differences in favor of the cognitive behavioral therapy intervention were noted for several secondary outcome measures as well (e.g., 6MWT, fatigue and daytime sleepiness scale, CIS-fatigue). Prior studies suggested that low to moderate intensity exercise was beneficial in DM1. While addition of graded exercise in a subset of the cognitive behavioral therapy group helped address fatigue and increased physical activity and participation (as evidenced by DM1-Activ-c), there was no improvement in self-reported quality of life. Adverse events and serious adverse events were approximately equal across these treatment groups—although study data indicated that efforts should be included to prevent falls when implementing the cognitive behavioral therapy paradigm into rehabilitation programs.

Taken together, the research team concluded that cognitive behavioral therapy alone resulted in improved capacity for activity and social participation in severely fatigued adult patients over the 10-month study period and stated that the approach could be considered in this group of DM1 patients. The knowledge and experience gained in the OPTIMISTIC trial experience should carry forward in the design, conduct, and analysis of future interventional clinical trials in DM1.

References:

Cognitive behavioural therapy with optional graded exercise therapy in patients with severe fatigue with myotonic dystrophy type 1: a multicentre, single-blind, randomised trial.
Okkersen K, Jimenez-Moreno C, Wenninger S, Daidj F, Glennon J, Cumming S, Littleford R, Monckton DG, Lochmüller H, Catt M, Faber CG, Hapca A, Donnan PT, Gorman G, Bassez G, Schoser B, Knoop H, Treweek S, van Engelen BGM; OPTIMISTIC consortium.
Lancet Neurol. 2018 Jun 18. pii: S1474-4422(18)30203-5. doi: 10.1016/S1474-4422(18)30203-5. [Epub ahead of print]

OPTIMISTIC Web Site
http://optimistic-dm.eu/

OPTIMISTIC ClinicalTrials.gov Entry
Observational Prolonged Trial in Myotonic Dystrophy Type 1 (OPTIMISTIC)
https://clinicaltrials.gov/ct2/show/NCT02118779

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 HSA^LR mouse skeletal muscle; both rescued multiple splicing events in HSA^LR mice; and both improved skeletal muscle histopathology and reduced myotonia in HSA^LR 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.

Synthesis

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.

References:

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.

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

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.

Reference:

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]

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 CCUG_exp 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 CCUG_exp, but not CUG_exp RNA

To assess whether alternative RNA binding proteins may be more efficient at binding to CCUG_exp versus CUG_exp, 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 CCUG_exp, but not CUG_exp. 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 CCUG_exp sequences—these studies confirmed a direct interaction between rbFOX1 and CCUG_exp.

rbFOX1 is Bound to CCUG_exp in DM2 Patient Cells

In additional experiments, the research team established that rbFOX1 co-localized with CCUG_exp–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 CCUG_exp 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 CCUG_exp, 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 CCUG_exp. 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 CCUG_exp 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 CCUG_exp versus CUG_exp repeats by rbFOX1 would mitigate the splicing defects and thereby potentially contribute towards the milder pathology in DM2.

Reference:

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.

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.

References:

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 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.

Reference:
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]

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.

References:

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.

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 info@myotonic.org