Insulin resistance has been recognized for decades as a common feature of type 1 myotonic dystrophy (DM1) and more recently of type 2 myotonic dystrophy (DM2), but it has yet to be fully understood or optimally treated.

Dramatic progress in understanding the molecular pathways underlying DM -- such as the knowledge that the DNA expansions in this disease cause abnormalities in RNA splicing for the insulin receptor -- has occurred in recent years. But there may be more to the insulin story in DM.

Searching for ‘Something More’

Laura Renna, Ph.D., of the IRCCS-Policlinico San Donato in Milan, Italy, has a 2016-1017 research fellowship from MDF and the UK-based Wyck Foundation to try to improve understanding and treatment of DM-related insulin resistance.

“We think that the alternative splicing of the insulin receptor is not the only reason behind insulin resistance in myotonic dystrophy patients,” Dr. Renna says. “We think there is something more. Our goal is to understand what this ‘something more’ is in DM skeletal muscle cells.”

Dr. Renna’s connection to the disease is personal, as her mother and brother are affected by DM1. “My mother has the late-onset form of the disease,” she says, “and my brother has the childhood phenotype and is very affected.” She received her Ph.D. in biomolecular sciences in November 2015, but her research focus has always been on DM.

Identifying Biomarkers, Testing Interventions

Dr. Renna’s research project, “A New Approach to Pathomolecular Mechanisms in Myotonic Dystrophy Insulin Resistance by Nutrigenomics,” will investigate the mechanisms that induce insulin resistance in DM patients and whether they contribute to weakness. 

The results are expected to lead to the identification of biomarkers that could become targets for therapeutic intervention. In addition, her research will test the ability of the natural insulin mimetics resveratrol, carnitine and betaine to modify insulin resistance and muscle atrophy in DM.

“Myotonic dystrophy patients are characterized by metabolic dysfunction, such as insulin resistance, hyperinsulinemia, and a high incidence of type 2 diabetes,” Dr. Renna says. “Insulin resistance represents one of the major abnormalities that can lead to cardiovascular disease, and cardiac manifestations are one of the most common features of myotonic dystrophy. Almost 30 percent of DM1 patients die from cardiac failure.”

Insulin resistance and its downstream effects aren’t different in DM patients compared to non-DM patients with this condition, she notes, but the molecular mechanisms that lead to it appear to be.

Dr. Renna and her colleagues will study insulin resistance and test these compounds in muscle biopsy samples derived from the biceps in patients with DM1, DM2 and healthy, age-matched controls, and in cultures of myoblasts and myotubes obtained from their satellite cells.

Special Treatments Needed for DM Patients 

A current first-line treatment for insulin resistance in patients with or without DM is the drug metformin [Glucophage], Renna says. “However,” she notes, “metformin can have long-term side effects, such as vitamin B12 deficiency, which can cause neuropathy. Moreover, some other effects have been observed, like liver and kidney alterations.” She says these could be particularly harmful in a multisystem disorder like DM1 or DM2, making the search for better treatments urgent.

Dietary modifications are recommended for DM and non-DM patients with insulin resistance, she notes, but many DM patients “need assistance in following dietary modifications” and may follow them only if the family is on board.

A New Approach with Insulin Mimetics

Dr. Renna’s team believes natural insulin mimetics may offer valuable new approaches to therapy. They will administer insulin mimetic compounds to the cells and then analyze the expression of proteins in the insulin pathway, as well as glucose uptake by the cells. 

“In the absence of these insulin mimetic compounds, DM muscle cells have lower glucose uptake,” she says. “We will analyze glucose uptake after administration of the natural compounds, comparing them with the administration of insulin or metformin.” The goals are to find biomolecular markers that can be used to measure therapeutic interventions and ultimately a cure for insulin resistance in myotonic dystrophy.

For betaine and carnitine, Renna says, there have been no trials in humans, but there have been tests on murine muscle cells in vitro.

“It seems they have a positive effect on the activation of the insulin pathway” in these cells, she says, and they will now test them in skeletal muscle cells taken from DM patients.

Resveratrol has been tested in patients with type 2 diabetes who do not have DM. However, it has mainly been tested as an adjunct to the usual therapy, and the trials have been limited to about 12 weeks. In some trials, with these caveats, the drug was helpful. “Additional trials are needed to see whether resveratrol or other insulin mimetic compounds can be used in the treatment of insulin resistance or diabetes,” Renna says.

Ginny Morriss, Ph.D., is exploring whether reducing levels of the CELF1 protein, which are abnormally high in DM1-affected skeletal muscles, has a positive effect on these muscles. She’ll be studying mice in which disease-causing repeat expansions can be induced at any age.

The expansion of CTG repeats in the DMPK gene on chromosome 19 has been understood to be the molecular cause of DM1 since the mid-1990s. More recently, studies from many laboratories around the world have shed light on the possible mechanisms by which these expanded repeats lead to skeletal muscle wasting and defects in multiple other body systems.

Two mechanisms that help explain DM1-related muscle wasting are the sequestration of MBNL1 protein by expanded CUG RNA repeats in skeletal muscle cells, with a resulting depletion of this critical splicing factor in these cells; and higher-than-normal levels of CELF1 protein in skeletal muscle cells, perhaps because its half-life is prolonged as yet another effect of expanded CUG RNA repeats.

The effects of MBNL1 depletion and the possible therapeutic effect of its replacement have been extensively explored in mouse studies. The effects of CELF1 overabundance and the possible therapeutic effect of its reduction are now receiving increasing scrutiny.

Exploring Muscle Development, Diseases

"Everything you do in life, whether you’re an elite athlete or just trying to walk up the stairs, requires muscles that function properly and grow in the right way," says Ginny Morriss, Ph.D., a postdoctoral associate in the Pathology Department at Baylor College of Medicine in Houston. "When I went for my postdoc, I was interested in muscle diseases, because there are defects in these diseases that involve some of the key developmental pathways in skeletal muscle. I got interested in the work that Dr. Tom Cooper was doing in his lab at Baylor. Not only were they studying myotonic dystrophy, but they were also delving pretty deeply into muscle development."

Dr. Morriss, now a postdoc in Dr. Cooper’s lab, has a 2016-2017 research fellowship from the UK-based Wyck Foundation in partnership with MDF to study the effects of abnormally high levels of CELF1 and of CELF1 reduction on skeletal muscles in an inducible mouse model of DM1.

"We can turn on the expanded repeats in these mice whenever we want," Morriss explains. "To mimic the congenital-onset type of DM1, we can turn them on very early, at the beginning of the postnatal period. Or we can wait until after the postnatal period of development and turn them on after that. We’re seeing clear signs of muscle wasting in these mice. What I’m proposing to do is start the expression of these repeats and then inject an adeno-associated virus with short hairpin RNA [shRNA] to CELF1 once the process of wasting has been established."

Normalizing MBNL1, CELF1 Levels

Morriss believes that reducing CELF1 levels and/or raising MBNL (muscleblind) protein levels have potential as DM1 therapies. "CELF1 overexpression and a decrease in muscleblind levels, primarily from muscleblind 1 and muscleblind 2 knockdown models, result in significant muscle wasting in mice," she says. "So restoring those proteins to the levels where they should be could be helpful." That’s something other investigators are working on, she notes.

She thinks it would probably be feasible to develop agents to increase MBNL1 and reduce CELF1 in patients. "Using adeno-associated virus [AAV] gene delivery is a relatively straightforward process that is being used extensively in a lot of clinical trials for other diseases," she says, noting that she counted 163 clinical trials that are now using AAV technology. "I think it is feasible to use AAV to put in muscleblind or to inject shRNAs to reduce CELF1. I think we may be able to deliver those to patients and target them pretty well. By the time we get there, a lot of the kinks will, I hope, have been worked out."

Morriss will use AAV9 to deliver shRNA to CELF1 to her mice. "It’s very highly specific for skeletal and cardiac muscle," she says, adding that reduced MBNL1 and CELF1 excess are relevant to heart as well as skeletal muscle. "For my studies, I’ll be injecting directly into the skeletal muscles, so there’s not a lot of chance of it getting up to the heart. But AAV9 also has the advantage of being good for systemic administration. If we wanted to use it for studying the heart phenotype in our models, we could."

Expecting to Stabilize Damage, Hoping to Reverse It

Asked whether targeting CELF1 with shRNA could actually reverse existing muscle damage, Morriss is cautious. "At this point, I’d probably say muscle damage would be closer to being stabilized than reversed. Until we know a little bit more about how well it can reverse damage in mice, we won’t know whether it might be possible to reverse it in patients. It might preserve muscle rather than reverse damage, but our goal is reversal."

Combining Therapies

Asked whether MBNL1 enhancement or CELF1 depletion or both could be useful in conjunction with therapies that target the CUG repeats, such as the antisense compound now being tested in patients, Morriss says she thinks they would be. "I think it would be helpful to have as many of these therapies as possible that are efficacious and safe, because there’s not going to be a one-size-fits-all treatment for this disease."

Heterogeneity in the presentation and course of DM1 is a well-recognized feature that has, thus far, complicated the management of patients and design of clinical trials. Gene discovery in other muscular dystrophies (e.g., CMD or LGMD) provided the means, not only for better classification, but of supporting patient care and therapy development. While we understand the relevant genes in DM, knowledge of modifiers of the DM1 clinical spectrum is lacking. In the absence of genetic or environmental disease modifiers, or of diagnostic or predictive biomarkers, the French Myotonic Dystrophy Clinical Network has evaluated a large cohort of DM1 patients in the DM-Scope registry and developed discrete disease profiles to support a five-grade model of DM1.

Although we understand the existence of heterogeneity in DM1 (differences in onset age, organ systems affected, and severity and order of appearance of symptoms), the curious dilemma for the field is what lies behind the substantial clinical variability. As yet, no modifier genes have been discovered that provide molecular links to DM1 molecular mechanisms and the observed heterogeneity, nor have any biomarkers been validated that can predict time of onset and severity of the multi-organ system consequences of DM1.

The French DM Clinical Network sought to better understand the heterogeneity of DM1, with the rationale that better characterized phenotypes would support improvements in mechanistic research, patient care, and clinical phases of drug development. Members of the Network recently published findings from a cohort of 2,167 adult DM1 patients evaluated across the 28 member neuromuscular centers.

Basing analysis on CTG repeat length and detailed evaluation of the occurrence, onset time and order of occurrence, and severity of symptoms, their data support a classification scheme with five types of DM1: congenital, infantile, juvenile, adult-onset, and late-onset. While CTG repeat lengths overlapped, the investigators showed differences in repeat length distribution among the five categories. CTG length alone did not appear to be a valid marker for disease prognosis.

The investigators established that co-varying patterns in CTG length distribution and age of onset and frequency of systems define five disease grades and suggest that patient care should be aligned with disease grade. Several of the clinical features of DM1 appeared to exhibit specific onset times that were linked to disease grade. Disease grade, for example, then could be used to predict the timing of need for specialty care, as the authors noted that cardiac and aging-associated features (e.g., cataracts, endocrine symptoms) can develop very early in specific DM1 grades. The classification scheme also appears to better refine understanding of patterns of symptomatology in childhood DM1.

Taken together, the five-grade classification system published by the French DM Research Network provides an important framework to guide patient care and, potentially, to stratify patients in interventional clinical trials. Clearly, taking the next step to better understand and track the mechanistic factors behind the heterogeneity in DM1 will be essential to characterization of patients, refinement of the five-grade model, and improvement of patient care and the design/stratification of clinical studies and trials.

Reference:

Unravelling the myotonic dystrophy type 1 clinical spectrum: A systematic registry-based study with implications for disease classification.
De Antonio M, Dogan C, Hamroun D, Mati M, Zerrouki S, Eymard B, Katsahian S, Bassez G.
French Myotonic Dystrophy Clinical Network. Rev Neurol (Paris). 2016 Sep 21. pii: S0035-3787(16)30205-3. doi: 10.1016/j.neurol.2016.08.003.

As potential new therapies for myotonic dystrophy (DM) progress through preclinical and clinical evaluation, drug developers need new tools to design and conduct clinical trials. Publically available tools to assess target engagement/modulation and dose-response in early-stage clinical trials would be particularly attractive to pharmaceutical and biotechnology companies that are either considering or in early stages of programs in DM.

A recent publication suggests that such a biomarker may be within reach. Drs. Andy Berglund, Eric Wang, Charles Thornton, and colleagues developed a doxycycline-inducible in vitro system to allow modulation of free MBNL protein concentration (free [MBNL]) across a 20-fold range. They used this system to establish that different splicing events require differing free [MBNL] and developed an algorithm to estimate free [MBNL] from splicing event data alone. The inducible system and Bayesian modeling allowed construction of dose-response curves (free [MBNL] vs. percent spliced in) and then used this system to evaluate the predictive value of any single splicing event and of splicing events in combination.

With the ability to determine the sensitivity of various splicing events, known to be affected in DM1, to infer free [MBNL], the research team applied this approach to tibialis anterior muscle biopsies from DM1 patients (44 DM1, 11 controls). Inferred free [MBNL] correlated well with both splicing dysregulation and disease status. Yet some splicing events were better than others in predicting free [MBNL] and some events functioned well only for particular levels of disease severity. The team then sought to determine which splicing events would be most informative as to free [MBNL] and DM1 patient status.

As expected, grouping study subjects by inferred free [MBNL] resulted in clustering of individuals with asymptomatic, proto-DM1 mutations with non-DM1 controls, since both cohorts lack MBNL sequestration. In more severe DM1, severity was linked to inferred free [MBNL], but the splicing events that conferred the best predictive power varied with disease severity. Combinations of splicing events were assessed to identify those that could report out across a wide dynamic range of free [MBNL] and disease severity. Using a cross-validation training/test set approach, splicing event combinations were selected that yielded optimal predictive power across the full spectrum of DM1 severity. Use of up to 30 splicing events in the panel improved accuracy and predictive power.

These data hold out the potential that a carefully selected panel of splicing events can accurately and reproducibly infer free [MBNL] across a wide dynamic range of both protein levels and disease severity. Such a panel could serve as an effective pharmacodynamics biomarker, providing an early signal that a candidate therapeutic was delivered to, engaged, and modulated the intended target. This biomarker would also have sufficient sensitivity for precise dose-response determinations in early stage clinical trials. Given the therapeutic strategies and modalities that are currently under study for DM, a biomarker that reads out free [MBNL] would have broad applicability.

While CUG repeat expansion in the DMPK gene is the proximate cause of DM1, repeat length only loosely correlates with age of onset and severity of disease, perhaps because it loosely correlates with non-sequestered MBNL. Accumulated research suggests that free [MBNL] is a direct determinant of the splicopathy thought to be responsible for the symptomatology of DM. Findings from this latest publication strongly suggest that we are on the cusp of qualifying a biomarker to accurately estimate free [MBNL], the target of many drug development programs in DM, and thereby implementing a powerful molecular tool into clinical assessment of patients and drug discovery and development programs.

Reference:

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.
Wagner SD, Struck AJ, Gupta R, Farnsworth DR, Mahady AE, Eichinger K, Thornton CA, Wang ET, Berglund JA.
PLoS Genet. 2016 Sep 28;12(9):e1006316.

Skeletal Muscle Transplants and the Pathobiology of Muscular Dystrophy

Transplantation of muscle precursor cells—to regenerate myofibers not compromised by the patient’s mutation--has attracted considerable attention as a candidate therapy for multiple forms of muscular dystrophy. While direct injection of stem cells into the target organ is feasible for some diseases, the sheer volume and body-wide distribution of skeletal muscle compromise direct injection strategies for muscular dystrophy. Translation to clinical trials using systemically delivered cells in humans then would face considerable difficulties including delivery, survival in the environment of dystrophic/regenerating muscle, and a host of regulatory issues regarding preparation, properties, and safety of a cell therapeutic. Unfortunately, the field is fraught with controversy as multiple international clinics offer stem cell ‘therapies’ of questionable efficacy and safety. Muscle transplant experiments in model organisms, however, may have value for identifying new aspects of the pathogenesis of muscular dystrophies, including myotonic dystrophy, and a recent study appears to have done that.

Intracellular Fate of Toxic RNA

A new study has utilized a novel mouse model, combining expression of pathogenic expanded CUG repeat RNA (HSA^LR) with an immunodeficient strain (NSG), thereby allowing study of muscle stem cell transplantation in a DM1 model (designated NSG-HSA^LR; Mondragon-Gonzalez et al., 2019). The research team’s findings may improve understanding of the trafficking of DM1-related toxic RNA within the unique environment of multinuclear skeletal myofibers.

Muscle progenitor cells (sourced from human iPAX7 PLZ iPS line, mouse satellite cells, or Pax3-inducible mES cells) were injected into pre-injured tibialis anterior muscles of NSG-HSA^LR mice, as well as native HSA^LR and NSG controls. Endpoint analyses (presence/absence of nuclear foci/MBNL sequestration and splicing patterns for selected transcripts known to be mis-spliced in HSA^LR) were performed 4 weeks after injection. Transplanted myonuclei could be identified by markers specific to each of the three cell sources.

The central finding of the study was identification of nuclear foci/MBNL sequestration in myonuclei of each category of transplanted muscle stem cells, indicating the translocation of toxic RNA transcripts from host myonuclei to mutation-free transplanted myonuclei. The team attributed this to toxic RNA released into and acquired from shared cytoplasm following formation of chimeric myofibers. RT-PCR using primers specific to transplanted human myoblasts showed DM1-related splicopathy in SERCA1, LDB3, and CACNA1S transcripts, thereby supporting the notion of internuclear transfer of toxic RNA. Controls fit expected patterns and were negative for nuclear foci and splicopathy.

Conclusions on the Mobility of Expanded Repeat RNA

Fusion of mutation-free donor precursor cells into existing NSG-HSA^LR myofibers was accompanied by translocation of toxic RNA, originating from host myonuclei, into donor myonuclei. This resulted in a DM1-like phenotype in donor cells. This finding suggests that (a) expanded repeat RNA is not confined to the nuclear domain it originated from and (b) toxic RNA then can migrate from cytoplasm into myonuclei other than where it originated, and result in formation of nuclear foci and the ensuing mis-splicing.

One implication of these findings, potentially broadly involving putative therapy development strategies in DM1, is that those myonuclei not exposed to a given therapy could spread pathology to treated myonuclei within the same myofiber and negate a positive effect. Thus, further exploration of the occurrence and mechanism of internuclear RNA transfer identified here is essential.

Reference:

Transplantation studies reveal internuclear transfer of toxic RNA in engrafted muscles of myotonic dystrophy 1 mice.
Mondragon-Gonzalez R, Azzag K, Selvaraj S, Yamamoto A, Perlingeiro RCR.
EBioMedicine. 2019 Aug 21. pii: S2352-3964(19)30553-5. doi: 10.1016/j.ebiom.2019.08.031. [Epub ahead of print]

New Review Article Series on DM

A special issue of the on-line International Journal of Molecular Sciences (edited by Prof. Lubov Timchenko) has been publishing a series of review articles on DM. To date, these articles have focused on the role of short tandem repeat expansions in RNA toxicity in DM1 and DM2 (Sznajder and Swanson, 2019) and on experiences with the development of CRISPR/Cas genome editing for DM1 (Raaijmakers et al., 2019). MDF's Research News recently highlighted one of these reviews. The latest piece in this series reviews small molecule drug development efforts aimed at DNA, RNA, and protein stages in the pathogenesis of DM1 (Reddy et al., 2019). The lead author of this review, Dr. Kaalak Reddy (University of Albany SUNY), is a former MDF Research Fellow.

Small Molecule Drugs for DM1

Small molecule compounds offer considerable advantages as putative, orally delivered drugs, a delivery route likely to be essential for systematically addressing multi-organ system diseases like DM. Knowledge of druggable chemical space (the depth and breadth of compounds with drug-like properties defined by Lipinski rule of 5 and beyond), and the analoging possible via medicinal chemistry, collectively allows: (a) high-throughput identification of parent compounds with activity at any one of multiple levels of the disease mechanisms operative in DM and (b) iterative compound optimization via analysis of Structure-Activity Relationships (SAR). Academic efforts toward discovery and development of small molecule drugs have improved in recent years, although considerable need for industry’s very large compound libraries, high-throughput capacity, and more rapid medicinal chemistry capability remains.

Dr. Reddy and colleagues frame their discussion around the molecular targets that are available to stem the pathogenesis of DM1, noting that much (but not all; e.g., AMO Pharma’s Tideglusib) progress has been made in targeting mechanisms downstream of either the expanded DNA repeats or toxic RNA. They proceed to document how that picture is changing.

The authors review, in detail, efforts for small molecule drug development for several targets/strategies, including targeting toxic RNA strategies based upon knowledge of target crystal structure (i.e., affinity for DM1 or DM2 expanded repeats), small molecule screens for toxic RNA targeting (including traditional screens, repurposed drug library screens, combinatorial chemistry screens, and specific target screens to disrupt toxic RNA-MBNL binding or nuclear foci), upregulation of MBNL protein, mis-spicing as a readout for high throughput screens, targeting CUGBP1, blocking toxic RNA transcription, targeting RAN translation, and modulating DNA expanded repeat instability. Taken together, the review serves as a digestible compendium of small molecule drug efforts in DM.

Potential for Small Molecule Drugs for DM

The authors have highlighted the breadth and depth of current efforts to bring candidate small molecule therapies into the clinic for DM. The potential for success is optimized by both the range of targets in the mainstream of established molecular mechanisms and the diversity of strategies applied to those targets. The oral bioavailability that can be achieved for small molecule drugs and their potential cost profile (versus recent pricing of biologics in other neuromuscular disease indications) also makes these efforts attractive. Finally, synergistic value may be obtained if two or more molecules receive marketing approval to address the primary pathogenic mechanisms in DM.

References:

Short Tandem Repeat Expansions and RNA-Mediated Pathogenesis in Myotonic Dystrophy.
Sznajder ŁJ, Swanson MS.
Int J Mol Sci. 2019 Jul 9;20(13). pii: E3365. doi: 10.3390/ijms20133365. Review.

CRISPR/Cas Applications in Myotonic Dystrophy: Expanding Opportunities.
Raaijmakers RHL, Ripken L, Ausems CRM, Wansink DG.
Int J Mol Sci. 2019 Jul 27;20(15). pii: E3689. doi: 10.3390/ijms20153689. Review.

Mitigating RNA Toxicity in Myotonic Dystrophy using Small Molecules.
Reddy K, Jenquin JR, Cleary JD, Berglund JA.
Int J Mol Sci. 2019 Aug 17;20(16). pii: E4017. doi: 10.3390/ijms20164017. Review.

The Problem of Clinical Outcome Measures in DM1

Despite much work being done to develop knowledge, clinical development tools, and relationships to de-risk the entire therapeutic development pipeline for DM1, the status of clinical trial outcome measures has remained a substantive hurdle. The intersection of the slow progression of most aspects of DM with the need for timely go/no decision-making on safety and efficacy of candidate therapeutics highlights the difficulties in arriving at a pragmatic battery of outcome measures. Moreover, the heterogeneity of presentation and progression of DM1 means that outcome measure development and validation can be driven only by sufficiently powered natural history studies using optimized operating procedures across multiple clinical sites.

Progress Toward DM1 Outcome Measures

Dr. Aura Cecilia Jimenez-Moreno (Newcastle University) and colleagues, working within the international Outcome Measures for Myotonic Dystrophy (OMMYD) group, have published a large cohort, cross-sectional and longitudinal analysis of a battery of tests aimed at identifying functional outcome measures for DM1 (Jimenez-Moreno et al., 2019). Measures included in the study were 6-minute walk test, 30-second sit and stand test, timed 10-minute walk test, timed 10-minute walk/run test, and 9-hole peg test, following specified operating procedures.

The OMMYD study was initiated with cross-sectional analysis of the five outcome measures in a cohort of 213 subjects recruited at two sites (University College London and Newcastle University; 98 subjects then were followed longitudinally at the NU site). The battery of tests selected here appears feasible for use in interventional clinical trials as 96% of the participants completed all outcome measures in a single test session. Both body mass index and disease severity associate with functional measures. Gender differences in outcome measure performance were a bit complex, with their relationship to established phenotypic variations in DM1 by gender (Dogan et al., 2016) unclear. Intra-session reliability analysis showed that two trials of each of the functional tests were sufficient to provide reliable/valid scores. Average scores of most functional tests significantly correlated with subject quantitative muscle strength, SARA score, and results of PROMs (MDHI and DM1-ActivC).

Longitudinal analysis of the Newcastle cohort involved a second follow-up at 12 months. This cohort included both adult- and late-onset phenotypic DM1. Except for the 9-hole peg test, all the functional tests and SARA showed significant changes from the first evaluation—changes were similar in both phenotypic groups.

Limitations and Path Forward

This study did not, however, report out on study subject perception of the meaningfulness of the functional changes in outcome measures (i.e., potential changes in burden of disease) detected across the 12-month longitudinal study period. This information will be vital to how regulatory agencies view the impact of a candidate therapeutic. The authors also note that use of independent evaluations on two successive days would have provided important data on instrument validity and measurement error. Assessment of inter-rater variations also was not determined. Finally, it was suggested that larger cohorts will be needed to account for differences in subgroups identifiable within the study cohort.

Overall, the OMMYD report makes many valuable specific recommendations for subsequent natural history studies geared toward assessing and validating clinical outcome measures for interventional trials in DM1. Continued efforts by OMMYD, the U.S.-based Myotonic Dystrophy Clinical Research Network, and other teams, particularly coordination among these efforts, may be essential to establish a validated protocol that is both feasible and sufficient for decision-making in clinical trials.

References:

Analysis of the functional capacity outcome measures for myotonic dystrophy.
Jimenez-Moreno AC, Nikolenko N, Kierkegaard M, Blain AP, Newman J, Massey C, Moat D, Sodhi J, Atalaia A, Gorman GS, Turner C, Lochmüller H.
Ann Clin Transl Neurol. 2019 Aug;6(8):1487-1497. doi: 10.1002/acn3.50845. Epub 2019 Jul 22.

Gender as a Modifying Factor Influencing Myotonic Dystrophy Type 1 Phenotype Severity and Mortality: A Nationwide Multiple Databases Cross-Sectional Observational Study.
Dogan C, De Antonio M, Hamroun D, Varet H, Fabbro M, Rougier F, Amarof K, Arne Bes MC, Bedat-Millet AL, Behin A, Bellance R, Bouhour F, Boutte C, Boyer F, Campana-Salort E, Chapon F, Cintas P, Desnuelle C, Deschamps R, Drouin-Garraud V, Ferrer X, Gervais-Bernard H, Ghorab K, Laforet P, Magot A, Magy L, Menard D, Minot MC, Nadaj-Pakleza A, Pellieux S, Pereon Y, Preudhomme M, Pouget J, Sacconi S, Sole G, Stojkovich T, Tiffreau V, Urtizberea A, Vial C, Zagnoli F, Caranhac G, Bourlier C, Riviere G, Geille A, Gherardi RK, Eymard B, Puymirat J, Katsahian S, Bassez G.
PLoS One. 2016 Feb 5;11(2):e0148264. doi: 10.1371/journal.pone.0148264. eCollection 2016.

As a consequence of the retention of mutant DMPK transcripts in the nucleus in DM1, patients express baseline levels of DMPK protein that are already half those of unaffected individuals. Since a key therapeutic strategy relies upon degradation of DMPK transcripts using antisense oligonucleotides (ASO), there are concerns as to whether essential functions of DMPK may be comprised and thereby contribute to the pathogenesis in DM1.

A University of Rochester team led by Dr. Charles Thornton has addressed this issue using mouse models with constitutive (genetic deletion) or acquired (ASO reduction) reductions in Dmpk. The function of DMPK is currently unknown. Prior reports in genetic models have shown that mice with heterozygous deletion of Dmpk exhibit cardiac conduction system defects, while those with homozygous deletion show skeletal muscle myopathy and weakness. Thus optimization of the ability of ASOs to target and degrade DMPK transcripts could exacerbate cardiac and skeletal muscle dysfunction in DM1.

Dr. Thornton and colleagues reevaluated the impact of genetic and ASO-induced reductions in Dmpk in two mouse models. They saw no effect of genetic deletion or ASO knockdown on cardiac (heart rate, PR interval, QRS duration, left ventricular contractile parameters) or skeletal (grip strength) functional measures, despite the substantial reductions that were achieved in Dmpk protein levels. Current strategies for ASO knockdown in DM1 utilize an allele-selective approach by targeting and degrading the mutant DMPK transcripts that are retained in the nucleus. However, there is the possibility that wild-type DMPK transcripts that traffic to the cytoplasm may also be degraded, as the next generation ASO chemistries result in more effective delivery to skeletal and cardiac muscles.

Yet despite the concern that substantial reductions in Dmpk protein may impact these muscles, Dr. Thornton’s team did not uncover any pathophysiology associated with Dmpk knockdown, even when genetic and ASO strategies were combined to yield as much as 90% reduction. Differences between the results of the Thornton team and prior investigations may relate to technical differences in the studies, mouse background strain differences, or features of the genetic knockout alleles.

The levels of Dmpk silencing seen in this study most likely would exceed those that could be obtained in DM1 patients, even with a highly effective ASO drug. The level of reduction of Dmpk in the mouse models also would likely exceed the reductions that are necessary to effectively restore the DM1-linked changes in mRNA splicing, and thereby mitigate DM1 signs and symptoms.

Hence these findings support the notion that strategies to increase the effectiveness of ASO candidate therapies can be effective in DM1 without increasing the risk of cardiac and skeletal muscle events.

Reference:

Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice.
Carrell ST, Carrell EM, Auerbach D, Pandey SK, Bennett CF, Dirksen RT, Thornton CA.
Hum Mol Genet. 2016 Aug 13. pii: ddw266.

Among the multisystem consequences of myotonic dystrophy, patients are at risk of renal dysfunction. A recent paper by Dr. Tsuyoshi Matsumura (National Hospital Organization Toneyama National Hospital, Japan) and colleagues evaluated circulating cystatin C (CysC) levels in patients with a variety of neuromuscular diseases and found the highest levels in DM1, potentially predictive of subsequent kidney damage and failure. These data stress that renal function monitoring, via CysC levels, should be an important component of care for individuals with DM1. The assessment of renal dysfunction in neuromuscular disease is complicated by the fact that creatinine, which increases in renal dysfunction, declines in parallel with skeletal muscle loss. By contrast, cystatin C (CysC) levels also represent an indicator of kidney function, but one that is not affected by changes variables such as muscle volume, food intake, or exercise.

Thus CysC, as well as the glomerular filtration rate of CysC (GFRcys), constitute superior biomarkers of kidney function when the disease process itself impacts serum creatinine levels. Dr. Matsumura and his colleagues retrospectively studied a cohort of 586 patients with neuromuscular disease (141 with DM1), using a variety of renal function measures to evaluate the relative risks for development of kidney disease. None of the subjects had signs of renal dysfunction at the time of analysis. After controlling for baseline age and gender differences, cross-disease comparisons showed that elevation in CysC and reduction in GFRcys were most pronounced in individuals with DM1, suggesting that they were at the highest risk for kidney failure.

The study also showed that there was a modest correlation between expanded CTG repeat length and CysC levels. Finally, the authors reported findings from two DM1 autopsies, where nephrosclerotic changes were observed relatively early in disease course  (both subjects in their 40’s); although CysC levels were not available for these subjects, these data support the need for further study of the natural history of kidney dysfunction in DM1, to determine best practices for patient care.

Elevated CysC has previously been reported in neuromuscular disease patients with renal dysfunction or failure, and has been suggested as a safety biomarker for interventional clinical trials in these diseases. Findings by Dr. Matsumura’s team suggest that DM1 patients may be at greater risk for kidney dysfunction and failure than those with other neuromuscular diseases. Moreover, CysC levels may be an important biomarker for careful management of DM, as well as for safety assessments of interventions in DM1 clinical trials.

Reference:

"Renal dysfunction can be a common complication in patients with myotonic dystrophy 1."
Matsumura T, Saito T, Yonemoto N, Nakamori M, Sugiura T, Nakamori A, Fujimura H, Sakoda S.
J Neurol Sci. 2016 Sep 15;368:266-71. doi: 10.1016/j.jns.2016.07.036. Epub 2016 Jul 15.

A recent study corroborated increased susceptibility to cancer in DM1, for women in particular, and linked the elevated risk to depressed levels of a tumor suppressor microRNA (miRNA). The association between DM1 and increased risk of certain types of cancer was first recognized in 1965. Recent studies have validated these initial findings and suggested that cancer risks in DM1 were greater in women, but the causative mechanisms remained unknown. 

Dr. Adolfo López de Munain (Donostia University Hospital, San Sebastián, Spain) and colleagues have now corroborated gender differences in susceptibility to cancer and identified potential molecular mechanisms behind the cancer risk in DM1. In a publication in Neurology, Dr. López de Munain’s team quantified cancer risk in a well-characterized cohort of 424 patients with DM1, representing > 18,000 patient years of data, and explored the potential molecular links between DM1 and cancer prevalence.

All patients in the study had molecular confirmation of DM1 and substantial longitudinal phenotypic data available. The observed numbers of cancers in the DM1 cohort were compared against the numbers that would be expected, as calculated from the Basque region’s overall prevalence numbers, in order to determine standardized cancer incidence ratios. The investigators also performed gene expression analyses as a first step to understand molecular mechanisms behind the cancer prevalence data.

When compared to a general, geographically controlled population, DM1 patients showed a 2-fold increased risk of developing cancer. Mean age of malignant cancer detection in the DM1 cohort was 47 years. Gastrointestinal, genitourinary, skin and thyroid were the most frequent sites for malignant tumors. Increased risk was stronger in women with DM1.

In the overall DM1 population evaluated in this study, cancer represented the third leading cause of death, after respiratory and circulatory diseases. Analyses of molecular factors that may differentiate the at-risk DM1 population included CTG repeat length and genome-wide expression analysis of blood leukocytes using Affymetrix microarrays. The authors did not find a correlation between expanded CTG repeat length and cancer risk. Genome wide expression analysis did show differential expression of several genes that were previously linked to cancer (e.g., PDK4, DAPK1, CASP5, and PLA2G7).

Moreover, female patients with DM1 displayed significant down-regulation of the miRNA-200c/141 tumor suppressor family, while levels of this miRNA were elevated in men with DM1. Prior studies, in non-DM cohorts, have shown an association between declines in miRNA-200c and tumor progression/poor prognosis. Although further studies will be needed to mechanistically link changes in the DM1 transcriptome to increased cancer risk, the data from the San Sebastián group supports a compelling hypothesis linking reduction in tumor suppressor genes to cancer risk in women with DM1. The gender-specific differences in susceptibility to cancer, and the linkage to reduced levels of miRNA-200c, are particularly compelling findings for validation in independent DM1 cohorts and further mechanistic analyses.

Reference:

"Cancer risk in DM1 is sex-related and linked to miRNA-200/141 downregulation."
Fernández-Torrón R, García-Puga M, Emparanza JI, Maneiro M, Cobo AM, Poza JJ, Espinal JB, Zulaica M, Ruiz I, Martorell L, Otaegui D, Matheu A, López de Munain A.
Neurology. 2016 Aug 24. pii: 10.1212/WNL.0000000000003124. [Epub ahead of print]