MDF’s Patient-Focused Drug Development (PFDD) meeting, held in 2016 in conjunction with the U.S. Food and Drug Administration (FDA), highlighted the critical role of patients in developing clinically-meaningful outcome measures to facilitate drug development and approval. Identification of the symptoms regarded as most important to patients and caregivers, and the benefits they would most hope to derive from a therapy, provides critical guidance that must be included from the earliest stages of drug development.

Patient registries, like MDF’s Myotonic Dystrophy Family Registry, the National Registry for Myotonic Dystrophy & Facioscapulohumeral Dystrophy at the University of Rochester, the DM-Scope Registry in France, and the UK Myotonic Dystrophy Patient Registry, represent critically important data repositories to facilitate studies of the burden of disease for those living with myotonic dystrophy (DM).

The UK DM Patient Registry published a cross-sectional analysis of self-reported data from 556 myotonic dystrophy type 1 (DM1) patients with a confirmed diagnosis, representing approximately 8.5% of the estimated affected population in the United Kingdom (UK). Registered patients were also able to nominate healthcare specialists to enter their genetic and clinical data; these data augmented patient profiles and served to validate the patient-reported registry format.

Registrant gender was equally distributed (51% female) and a positive family history of DM was reported by 89% of registrants. Mean age of onset was 33 years. Fatigue/daytime sleepiness (79%) and myotonia (78%) were the most frequently reported symptoms—the occurrence of myotonia positively correlated with fatigue, dysphagia and ambulatory status. As in a prior report by the DM-Scope Registry, the UK cohort showed a higher frequency of severe myotonia in males, although fatigue did not show gender bias. Men also reported a higher frequency of cardiac abnormalities, non-invasive ventilation, and mobility impairments, while cataract surgery was more common in women. Most patients (65%) did not require assistive devices to walk. Severity of symptoms did not correlate with CTG repeat length obtained at the time of genetic testing.

While only one-third of patients reported having EKG, 48% of those were diagnosed with a cardiac conduction system abnormality and 36% had received an implanted cardiac device. Non-invasive ventilation was used regularly by 15% of patients. Of the patients with data available, 26% reported cataract surgery.

The UK group reported that the delays in receiving a genetic diagnosis of DM1 remain substantial and do not seem to have improved since the advent of genetic testing in 1996. They stress the importance of reducing the genetic diagnostic odyssey, not only to improve patient care and quality of life, but also to facilitate community readiness for interventional trials and approved therapies.

Taken together, knowledge of the symptoms that are most important to DM1 patients provides guidance not only for improved care, but also for the development of novel therapeutics. Studies such as that reported here, from the UK registry, provide an evidence-based underpinning that is essential for progress. An improved collaborative environment, whereby registry data is more readily shared, is a goal that will not only improve the science, but is in the best interests of those living with DM.

Reference:

The UK Myotonic Dystrophy Patient Registry: Facilitating and Accelerating Clinical Research
Wood L, Cordts I, Atalaia A, Marini-Bettolo C, Maddison P, Phillips M, Roberts M, Rogers M, Hammans S, Straub V, Petty R, Orrell R, Monckton DG, Nikolenko N, Jimenez-Moreno AC, Thompson R, Hilton-Jones D, Turner C, Lochmüller H.
J Neurol. 2017 Apr 10. doi: 10.1007/s00415-017-8483-2. [Epub ahead of print]

The burden of disease for myotonic dystrophy (DM) is multi-systemic, including skeletal muscle weakness, fatigue, cardiac arrhythmias, respiratory insufficiency and intellectual disability. Modeling of this constellation of symptoms is important for mechanistic studies and preclinical therapy development. Yet, what is arguably the most widely available mouse model, the HSA-LR, is designed to model the skeletal muscle molecular, structural and functional deficits of skeletal muscle.

Dr. Guey-Shin Wang and colleagues at Academia Sinica (Taiwan) developed an EpA960/CaMKII-Cre mouse that carries an inducible human DMPK with 960 CTG repeats in the 3’ UTR. Cre expression occurs in the forebrain (cortex and hippocampus) after two weeks of age, preserving earlier neurodevelopmental events from toxic RNA exposure and MBNL depletion.

Atrophy of the cortex and corpus callosum, features of the myotonic dystrophy type 1 (DM1) patient central nervous system (CNS), were observed in the adult EpA960/CaMKII-Cre mice. The mice showed progressive neuropathological findings: reduced MBNL1 in dendrites of cortical layer V neurons at six months of age, brain atrophy (axon and dendrite pathology) by nine months, and loss of MBNL2 and MBNL-directed splicopathy were later events, at 12 months.

Functionally, EpA960/CaMKII-Cre mice were evaluated at six months for hippocampus-dependent spatial learning and memory using the Morris water maze—escape latencies were greater for mutants compared with controls, suggestive of slower learning. Evaluation of long-term potentiation (LTP) in hippocampal slices at six months of age also was consistent with synaptic dysfunction and the functional learning disabilities seen in these mice.

The EpA960/CaMKII-Cre mouse model recapitulates some features, including disease progression, seen in DM1 patient CNS. Moreover, the authors suggest that depletion of MBNL1 and MBNL2 in the CNS are distinct events with differing roles in disease progression. Early synaptic dysfunction may be triggered by MBNL1 depletion, and is a precursor to dendritic and axonal pathology. Since splicing changes were detected only later, commensurate with MBNL2 depletion, the early synaptic dysfunction likely is mediated by MBNL1-related, but alternative splicing-independent events.

Reference:

Reduced Cytoplasmic MBNL1 is an Early Event in a Brain-specific Mouse Model of Myotonic Dystrophy.
Wang PY, Lin YM, Wang LH, Kuo TY, Cheng SJ, Wang GS.
Hum Mol Genet. 2017 Mar 24. doi: 10.1093/hmg/ddx115.

Inheritance of congenital myotonic dystrophy (CDM) is almost exclusively maternal and, while typically associated with large CTG expansions, is not always genetically differentiated from myotonic dystrophy type 1 (DM1) by repeat tract length. Correlations between CDM/DM1 genotype and phenotype can be improved through evaluation of somatic expansions. Yet it is clear that factors other than germ line repeat length underlie the bias toward maternal inheritance and the heterogeneity of CDM.

The laboratories of Drs. Karen Sermon (Vrije Universiteit Brussel) and Chris Pearson (Hospital for Sick Children) recently collaborated on an epigenetic analysis of the DM1 genetic locus in a cohort of DM1 and CDM patients. Prior reports showed that the DM1 locus resides in a 3.5 kB CpG island with putative CTCF sites, suggesting an epigenetic mechanism for DM1 regulation and disease phenotypes that diverge from CTG length assessments. Earlier reports also established variability in methylation status at that locus in both DM1 patients and DM1 transgenic mice. Drs. Sermon and Pearson hypothesized that CTG expansion might alter CpG methylation status and that a consequent regulatory dysfunction contributes to the severity of the CDM phenotype.

Drs. Sermon and Pearson and team evaluated multiple generations of several families, including 20 individuals with CDM. Results showed nearly an absolute correlation between the methylation status upstream of the expanded CTG repeat and the occurrence of CDM (19/20 cases). By contrast, this pattern of methylation was rarely found among DM1 patients (2/59 cases). The authors suggest that CpG site methylation is an important contributing factor, with the development of CDM not being determined by CTG repeat length alone.

Analysis of human embryonic stem cells (hESC) and chorionic villus samples from the study cohort identified upstream CpG site methylation only in maternally-derived samples; paternal samples never showed methylation upstream of expanded DMPK alleles.

Generational increases in both methylation and CTG expansion length were seen in each CDM family studied. Yet since CTG repeat lengths overlapped in DM1 and CDM, while upstream methylation was almost exclusive to CDM, the authors concluded that methylation status is a stronger indicator of CDM than absolute repeat length. Moreover, they speculate that the maternal inheritance bias of CDM may be a consequence of a failed survival of spermatogonia carrying the pathogenic methylation upstream of DMPK. Importantly, while their data suggests that it is rare, the authors do not exclude paternal inheritance for CDM.

Reference:

CpG Methylation, a Parent-of-Origin Effect for Maternal-Biased Transmission of Congenital Myotonic Dystrophy.
Barbé L, Lanni S, López-Castel A, Franck S, Spits C, Keymolen K, Seneca S, Tomé S, Miron I, Letourneau J, Liang M, Choufani S, Weksberg R, Wilson MD, Sedlacek Z, Gagnon C, Musova Z, Chitayat D, Shannon P, Mathieu J, Sermon K, Pearson CE.
Am J Hum Genet. 2017 Mar 2;100(3):488-505. doi: 10.1016/j.ajhg.2017.01.033.

Cardiac troponin-I, a sarcomeric regulatory protein integral to skeletal and cardiac muscle contraction, has long been utilized as a diagnostic and prognostic biomarker of heart disease. For muscular dystrophies, elevated serum creatine kinase and troponin are associated with myopathic changes in muscle. Understanding the sensitivity of the analytical tools, as well as the types of cardiac issues that may result in elevated cardiac markers in serum, is critical to use of these assays in monitoring myotonic dystrophy type 1 (DM1) patients.

The constellation of cardiac involvement in DM1 includes atrioventricular block, prolonged QT interval, prolonged QRS interval, increased ventricular premature contractions, atrial fibrillation/flutter, right/left bundle branch block, non-sustained ventricular tachycardia and left ventricular systolic dysfunction (Petri et al., Int. J. Cardiol. 160: 82-88, 2012). Prior reports identified a correlation between CTG repeat length and cardiac dysfunction and linked the degree of neuromuscular and cardiac involvement in patients.

A large multi-center study in Scotland recently reported out an analysis of serum levels of cardiac troponin-I (cTnI) in a cohort of 117 well-characterized DM1 patients recruited from outpatient clinics. Nine subjects had cTnI levels that exceeded the 99th percentile of the general population. One-third of subjects with elevated cTnI also had left ventricular systolic dysfunction. The authors noted that elevations in cTnI did not correlate with CTG length, were not predictive of severe conduction abnormalities and did not correlate with muscle strength (by MIRS score). There also was no association between cTnI level and the presence of an implanted cardiac device.

Overall, the authors suggest that cTnI levels represent a potential biomarker to assess risks in the management of DM1 patients and for stratification of subjects in clinical trials. Although the lack of correlation of cTnI levels and MIRS score suggests a cardiac origin for elevated serum cTnI, the underlying responsible pathology in the context of known cardiac phenotype of DM1 is currently unclear. Finally, the authors suggest that the overall sample of patients with elevated cTnI is small and propose these findings as exploratory, requiring follow-up of this and other putative cardiac biomarkers in larger cohorts.

Reference:

Elevated Plasma Levels of Cardiac Troponin-I Predict Left Ventricular Systolic Dysfunction in Patients with Myotonic Dystrophy Type 1: A Multicentre Cohort Follow-up Study.
Hamilton MJ, Robb Y, Cumming S, Gregory H, Duncan A, Rahman M, McKeown A, McWilliam C, Dean J, Wilcox A, Farrugia ME, Cooper A, McGhie J, Adam B, Petty R; Scottish Myotonic Dystrophy Consortium., Longman C, Findlay I, Japp A, Monckton DG, Denvir MA.
PLoS One. 2017 Mar 21;12(3):e0174166. doi: 10.1371/journal.pone.0174166.

A potentially revolutionary technology may allow development of a drug for DM that can correct a patient’s DNA by selectively removing the expanded CTG and CCTG repeats in DM1 and DM2, respectively.

This new gene editing technology has emerged from the discovery of how bacteria protect themselves from invading viruses. There very likely will be a Nobel Prize awarded to scientists who discovered how this bacterial defense mechanism could be used to edit human gene defects. There certainly has been a rather public fight over the patent rights that are potentially worth billions of dollars among the research groups at the University of California, Berkeley and the Eli and Edythe L. Broad Institute of MIT and Harvard.

CRISPR is an acronym for short, repetitive DNA sequences that function to immunize bacteria from viruses. CRISPR DNA works together with a family of proteins, known as Cas proteins, which cut and thereby inactivate the invading virus’ DNA. Working together, CRISPR and Cas can recognize viral DNA as foreign and then inactivate it via the DNA-cutting Cas protein.

Researchers Jennifer Doudna (U.C. Berkeley) and Feng Zhang (Broad) have shown that the CRISPR/Cas system can be adapted to cut human DNA at highly specified locations to either remove existing genes or to insert new genes. The potential value for DM is that known mutation-containing DNA sequences, specifically the expanded repeats in DMPK or CNBP, could be cut to remove the disease-causing expanded repeats. Doudna and Zhang are likely to share a Nobel Prize for this discovery, while it appears that Zhang and the Broad Institute have won the patent rights battle.

Is gene editing using the CRISPR/Cas system going to be available soon for DM? The direct answer is, no. Several issues need to be resolved before any clinical application of gene editing is realized. The specificity and efficiency of gene editing will need to improve. Delivery of the gene editing reagents also must be optimized—these are large molecules that will have to be delivered by intravenous injections and must then gain access to cells throughout the body of DM patients in order to correct the multiple symptoms of the disease. Lastly, there is the issue of safety—studies need to show not only that the CRISPR/Cas system is designed to edit the DMPK or CNBP genes, but that it does not cause harm by editing unintended genes. MDF is working with researchers and biotechnology companies to help advance CRISPR/Cas gene editing for DM.

Gene editing is entering clinical trials this year for some types of cancer. The strategy is to edit the patient’s immune cells, outside of their body (thereby circumventing some barriers to the technology), and the cells that are put back into the body have gene edits so they attack the tumor. This human study is an important proof of concept, and a step that will be critical as we move toward the application of gene editing for DM.

Gene Editing

The basic science discovery of an adaptive immunity system that evolved in bacteria as a DNA-targeting viral defense mechanism was so revolutionary that early manuscripts from three independent labs were rejected by multiple journals. From that inauspicious beginning, CRISPR/Cas9 technology is rapidly advancing toward clinical trials in multiple disease indications. 

Even that renowned purveyor of scientific knowledge, The New Yorker, has touted the therapeutic potential of CRISPR/Cas9 gene editing (see "The Gene Hackers").

Gene editing is rapidly moving toward clinical trials, indeed ex vivo editing of T-cells in order to target tumors was approved by the National Institutes of Health’s (NIH) Recombinant DNA Advisory Committee (RAC) and trials are anticipated to start in early 2017. By design, the oncology clinical trial largely avoids many potential barriers to CRISPR/Cas9-based therapies—efficiency of gene editing, safety, delivery and ethics. It is not surprising that a disease where ex vivo gene editing is a plausible therapeutic strategy is the first to reach clinical trials.

Possibilities for DM

For myotonic dystrophy (DM), gene editing is an attractive, but currently theoretical strategy for directly addressing the primary genetic defect by excising pathogenic expanded CTG or CTTG repeats. Recognizing that expanded repeats are present in every cell, thereby requiring in vivo gene editing, the DM field must address all of the barriers noted above if clinical trials are to become a reality.

A recent publication from Dr. Bé Wieringa and colleagues takes an important step in assessing the feasibility of CRISPR/Cas9-mediated somatic gene editing for DM. In studies of myoblasts from normal subjects, DM1 patients, and immortalized mouse myoblasts (DM500), the research team explored ways of modulating efficacy of expanded repeat excision. They evaluated both unilateral (from one side of the repeat tract) and dual CRISPR cleavage strategies. 

The group’s findings show specificity in removal of normal and expanded CTG repeats from the DMPK locus. Their dual CRISPR editing approach resulted in unusually large deletions (kilobase size, encompassing the entire expanded repeat) with no adverse biologic consequences for gene expression, DMPK mRNA localization, MBNL distribution or myogenesis. Unilateral cleavage of an unstable genomic repeat was viewed as unadvised, since the ensuing recombination repair may produce unpredictable genomic changes.

The promise of gene editing lies in its objective of stopping downstream pathogenic mechanisms via correction of the primary DNA defect. The challenges lie in establishing safety (putative off-target editing), further optimization of editing efficacy/efficiency, and ensuring bioavailability of CRISPR/Cas9 reagents to tissues impacted by DM. These are not trivial barriers, but the latest findings by Dr. Wieringa and colleagues provide important in vitro proof of concept and thereby represent an important step.

Reference:

CRISPR/Cas9-Induced (CTG⋅CAG)n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing
van Agtmaal EL, André LM, Willemse M, Cumming SA, van Kessel ID, van den Broek WJ, Gourdon G, Furling D, Mouly V, Monckton DG, Wansink DG, Wieringa B.
Mol Ther. 2017 Jan 4;25(1):24-43. doi: 10.1016/j.ymthe. 2016.10.014.

When Dr. Thurman Wheeler was a resident in neurology, he remembers a senior physician telling him that myotonic dystrophy would probably be one of the most difficult diseases to treat because it involves so many body systems. But thanks to unprecedented advances in laboratory and clinical research since then, “it looks like it might turn out to be fairly straightforward,” Dr. Wheeler says. Now an assistant professor of neurology at Harvard Medical School and a clinical neurologist at Massachusetts General Hospital, Wheeler has spent more than a decade caring for patients with myotonic dystrophy (DM) and conducting lab-based DM studies using mouse models.

Dr. Wheeler recently received a one-year grant through MDF to develop new serum-based biomarkers in adults and children with type 1 and 2 myotonic dystrophy (DM1 and DM2) for use in therapeutic trials. (For more about MDF grants, see Fellows & Grant Recipients. Additionally, information about the Wyck Foundation and its related grantees is available).

Searching for DM Biomarkers in Body Fluids

Dr. Wheeler’s grant, which runs from November 2016 through October 2017, will allow him and his team to begin initial exploration of the viability of developing DM biomarkers that can be measured in blood and urine, reducing or avoiding the need for muscle biopsies – which are invasive and risky – to support data collection in clinical studies and trials.

Dr. Wheeler will examine differences in extracellular RNA that are associated with DM1 and DM2 compared with healthy controls, and look for possible changes in these RNA forms and levels that correlate with disease activity or treatment response.

"We’re looking for extracellular RNA in blood and urine," Wheeler says. "A few years ago, a colleague here found that blood has extracellular RNAs that can serve as biomarkers for brain tumors," Wheeler says. "We’re going to be adapting the approach of the study that looked for markers of brain tumors and use that for myotonic dystrophy. We’re examining gene expression, splicing, microRNAs, and things like that."

Dr. Wheeler and colleagues will be studying extracellular RNA in adults and children with DM1 and DM2, in collaboration with neurologist Basil Darras at Boston Children’s Hospital.

The collaboration with Dr. Darras, who sees more pediatric patients than does Dr. Wheeler, is important, Wheeler says, “because this enables us to expand the study in children. Muscle biopsies in children require general anesthesia, he notes, “and that’s something you want to avoid in myotonic dystrophy, because patients can have a difficult time coming out of it. So, if we’re successful, we may be able to include children [in clinical trials] much earlier than originally thought.”

Early Years in the Clinic and Lab

Wheeler, who graduated from the University of Washington School of Medicine in 1995 and then completed a neurology residency at that institution, first became interested in muscular dystrophy research during a fellowship in neuromuscular medicine at Johns Hopkins University.

He then moved to Stanford University to work with Tom Rando, M.D., Ph.D., on developing nonviral gene therapy for Duchenne muscular dystrophy. Then, as now, the potential for unwanted effects associated with using viral vectors as gene delivery vehicles was well understood, and the Rando lab was looking to reduce this downside.

"We were doing plasmid and oligo work," Wheeler recalls. "[Dr. Rando] was using a type of non-viral gene therapy called antisense for gene correction of point mutations in a mouse model of Duchenne muscular dystrophy.  It involves using an oligo that’s complementary to the region across the point mutation except it has the correct base."

After three years at Stanford, Wheeler took advantage of an opening at the University of Rochester (N.Y.) to switch gears and study DM. “I knew what myotonic dystrophy was," he says, “but I had never done any research on it. I moved to Rochester, took what I learned about nonviral gene therapy from Tom, and applied it to myotonic dystrophy.”

"We ended up getting antisense to work for exon skipping to eliminate myotonia [in a DM mouse model]. The chloride channel RNA is misspliced in myotonic dystrophy. There’s an exon included aberrantly in the disease state. So if you use antisense to induce skipping of that exon, that can potentially rescue the myotonia, because you’d be restoring the normal chloride channel RNA, and that leads to a normal chloride channel protein. We did that in mice, and it worked beautifully.”

Correction of chloride channel splicing wasn’t taken forward into drug development, Wheeler says, "because there’s much more to myotonic dystrophy than myotonia. You’d eliminate the myotonia, but ultimately you’d be doing nothing for the rest of the symptoms."

It did, however, provide evidence that antisense could be an effective therapy, setting the stage for therapy development to target the fundamental DM1 RNA defect – expanded CUG repeats in the DMPK gene. “In parallel, we were working on CUG targeting,” Wheeler says of his Rochester work. “We were doing them both at the same time, and we finished the chloride channel work first. But we knew that the CUG targeting was working in the mice and that that could be a long-term answer.”

Developing Antisense for DM1 Treatment 

At first, the Rochester team’s goal was to develop antisense against the CUG repeat expansion in the DMPK gene. "We originally were using antisense that was targeting the repeat expansion directly," Wheeler recalls, "and the concern was that there are other genes that have shorter CUG repeats where that could interfere. We didn’t really find that in mice, but it was a theoretical concern."

Then came involvement with Ionis Pharmaceuticals, a Carlsbad, CA-based biotechnology company specializing in RNA-targeted drug discovery and development. 

"We tried some of the Ionis drugs that they developed earlier, but they didn’t really work very well," Wheeler says. "Then Ionis suggested we try their gapmer approach, and that worked incredibly well." A gapmer, he explains, refers to the design of the antisense. “The antisense has modified RNA at the 3-prime and 5-prime ends, separated by a central gap of DNA. When the oligo binds to the target RNA, you get a DNA-RNA heteroduplex that is recognized by RNAse H, which cleaves it. When the cleavage happens, the rest of the transcript is degraded by exonucleases." Non-gapmer antisense compounds, he explains, "just bind to the target and kind of sit there."

Unlike earlier antisense approaches for DM1, the Ionis gapmer approach did not directly target the CUG repeat expansion. "It targets outside the repeats," Wheeler says, thus removing the risk of inadvertent binding to CUG repeats in other genes. And, since RNAse H is located in the nucleus, the strategy preferentially targets aberrant DMPK RNA transcripts, which get stuck in that location, while normal DMPK RNA quickly leaves the area. "Transcripts that have a prolonged dwell time in the nucleus appear to be more susceptible,” he notes, although “potentially, the gapmer still could target the pre-messenger RNA of DMPK alleles with non-expanded repeats [normal alleles], so that is one of the things we’ll be watching in the clinical trials."

A phase 1-2 trial of IONIS-DMPKRx-2.5 in adults with DM1, testing the gapmer antisense against DMPK RNA at multiple U.S. centers, opened in 2014. It ended in late 2016, with results reported in January 2017. “I know that Ionis has taken great steps to test the safety ahead of time, and it’s been very effective in mice and other preclinical models of DM1,” Wheeler says. While the Ionis clinical trial did not achieve sufficient drug levels in skeletal muscle, they are exploring two other antisense oligonucleotide molecules that show promise of greater potency.

Move to Harvard

As fruitful and exciting as his time at the University of Rochester was, Wheeler was eager to expand his lab-based research and begin clinical work in DM and other muscular dystrophies. With that in mind, in 2013, he relocated to Massachusetts General Hospital and Harvard Medical School.  “It was just a great professional opportunity,” he says. “It was a natural step. In Rochester, I was doing no clinical work. Here I have a research lab that focuses on developing new biomarkers, including this new clinical project [for biomarker identification], as well as studying the factors that make muscles weaker and identifying new treatments for myotonic dystrophy. I also have an all-ages clinic every week and a pediatric clinic twice a month where I see patients with both types of myotonic dystrophy and all other forms of muscular dystrophy.”

Improving and Expanding Clinical Trials

“I’m optimistic that [antisense oligonucleotide therapies] will be safe and have some therapeutic effects,” Wheeler says. “I guess the question is to what extent the knockdown of the expanded repeat RNA will reverse the symptoms. In someone with mild symptoms, the drug may have a tremendous effect and slow progression.  But how will it work for patients who have a greater degree of weakness, more muscle atrophy, or problems walking?  Will the drug be able to improve their function at all? Or will we need to develop second-line therapies, the way the Duchenne dystrophy field is doing?”

Downstream effects of the expanded CUG repeats that appear to contribute significantly to disease symptoms include functionally low levels of the MBNL proteins and abnormally high levels of the CELF1 protein. Increasing MBNL activity and reducing total CELF1, preferably with small molecules, might add a lot to antisense therapy, Wheeler notes. “A small molecule that you could take by mouth would be ideal,” he says. “Until we have something that is proven to be highly effective, I think we should continue developing new therapies that target the disease from different angles.”

Continuing clinical trials of new DM therapies, including those for children, will require reliable biomarkers of disease activity, preferably markers accessible in blood or urine rather than muscle markers that require biopsies. 

“The plan is to begin the process of identifying biomarkers,” Wheeler says of his new grant. “That was one of the goals described by MDF. They want to find a project that is working toward biomarkers that will be on track to get qualified by the Food and Drug Administration (FDA).  

“The goal that I have is to try and reduce the need for muscle biopsies by looking at biofluids, so that there’s no anesthesia, no incision, no scarring, and no bleeding risk. This would allow monitoring during the treatment trial instead of waiting until the end.

“We had our first contact with the FDA back when the grant was submitted, which was in June [2016]. I think that no one expects that we’re going to have a biomarker or a group of biomarkers by the end of the year, but we’re going to be working toward that goal.”

For more about Dr. Wheeler’s MGH-based research, see Wheeler Muscular Dystrophy Research Lab. For information about muscular dystrophy clinics for adults, call (617) 726-3642; for children, call (617) 643-4645. Recruitment for the biomarker study is being done through the clinics.

Patient-reported data from MDF’s Myotonic Dystrophy Family Registry (MDFR) indicate that impaired sleep or daytime sleepiness are among the most prevalent symptoms of DM1, experienced by over 76% of patients. Despite the prevalence and substantial burden imposed by sleep disorders, studies report that many patients do not receive treatment.

Dr. Sophie West and colleagues at the Newcastle Regional Sleep Service and Institute of Genetic Medicine, Newcastle University have evaluated a large cohort of patients with daytime sleepiness and assessed the effectiveness of a variety of treatment strategies. Since many DM1 patients do not have immediate access to facilities providing optimized and integrated care, it is important to understand and disseminate best care practices among their physicians.

In a prospective study of 120 DM1 individuals presenting with daytime sleepiness, the Newcastle group used thorough overnight sleep studies to stratify patients into treatment regimens that are commonly used for sleep disorders but not yet rigorously validated for DM. Obstructive sleep apnea, respiratory failure, and sleepiness accompanied by a normal sleep study were prevalent among the DM1 study patients. Four treatment regimens were evaluated: (a) those with hypercapnia were offered non-invasive ventilation (NIV); (b) obstructive sleep apnea patients offered continuous positive airway pressure (CPAP); (c) those with daytime sleepiness and no underlying disorder detected from sleep studies were offered modafinil; and (d) a non-treatment group with normal sleep studies or with disorders but declined the interventions.

Those receiving each intervention and, parenthetically, the percentage deriving benefit were as follows: NIV for respiratory failure (37%), CPAP for obstructive sleep apnea (33%), and modafinil for daytime sleepiness with no sleep disorder (33%). Overall, 29% of patients studied derived benefit from interventions that are in general use for more common disorders. Differences in Epworth Sleepiness Score distinguished responders (ESS = 15.9) from non-responders (ESS = 11.9) across all interventions. The authors point to the need for either a meta-analysis of published studies or a randomized controlled trial in order to better understand the features of modafinil non-responders.

Report Findings

Dr. West and colleagues conclude that comprehensive diagnosis of sleep disorders in DM1 is essential in directing patients toward specific, most-effective interventions. They point to the diverse causes of sleep and breathing disorders in DM and advocate for this tailored approach to treatment. Taken together, the authors view a detailed, diagnostic approach as superior, both in treatment efficacy and overall costs, to simply using the predominant symptomatology at presentation as a guide to improving patient care. These findings also provide a strong argument for comprehensive care guidelines, readily available to both physicians and patients, to ensure optimum quality of care for all living with DM.

Reference:

Sleepiness and Sleep-related Breathing Disorders in Myotonic Dystrophy and Responses to Treatment: A Prospective Cohort Study.
West SD, Lochmüller H, Hughes J, Atalaia A, Marini-Bettolo C, Baudouin SV, Anderson KN.
J Neuromuscul Dis. 2016 Nov 29;3(4):529-537

Construction of a conceptual framework that integrates phenotypic, cellular and molecular data in DM is a critical step in developing a robust and diverse pipeline of candidate therapies. Although basic science has mechanistically linked the inherited repeat expansions to DM1 and DM2 phenotypes, there are critical gaps in understanding of disease mechanisms. A recent publication extends our understanding.

Dr. Perrine Castets, Prof. Michael Sinnreich and colleagues at the University of Basel recently studied the notion that perturbation of skeletal muscle metabolic pathways, including those responsible for protein degradation (ubiquitin-proteasome system and autophagy), plays an important role in DM. Their results, published in the Journal of Clinical Investigation, establish that (a) DM1 muscle is characterized by an altered response to energy/nutrient deprivation and that (b) dysregulation of AMPK/mTORC1 signaling, at least in part, underlies the altered metabolic state and its role in the pathogenesis of DM1 skeletal muscle. Importantly, these findings suggest new targets for drug discovery and development.

In studies of the HSALR mouse model of DM1, the investigators showed that a normal molecular response to fasting, AMPK activation and mTORC1 inhibition, is compromised in HSALR mice. Consistent with these findings and the interrelated role of AMPK and mTORC1 in autophagy, experimentally induced autophagy was disrupted in HSALR muscle. Deprivation of energy and nutrient supply in DM1 patient myotubes also produced data consistent with dysregulated autophagy. Finally, targeting either AMPK (with AICAR) or mTORC1 (with rapamycin) signaling improved muscle strength, splicing and/or myotonia in HSALR mice.

While the AMPK agonist, AICAR, disrupted nuclear foci and reduced myotonia, along with partial normalization of splicing (correction of Clcn1, but not Atp2a1 and Camk2b) in HSALR mice, rapamycin’s, an mTORC1 inhibitor, normalization of muscle function was not accompanied by correction of mis-splicing.

Many of the therapeutic strategies under development for DM are based on restoration of dysregulated alternative splicing. The study by the University of Basel group further supports those strategies, but also characterizes a key metabolic defect in DM1 muscle and identifies the mTORC1 pathway as an alternative, splicing-independent target for therapy development.

Thus far, nearly all of the therapy development programs in DM address the muscle phenotype. Recent studies show that the mTOR pathway may be an important target in developmental intellectual disorders, and two mTOR inhibitors have regulatory approval for other indications (Novartis' Everolimus and Wyeth's Sirolimus (rapamycin)). Drugs targeting mTORC1 then may have efficacy for both the skeletal muscle and cognitive symptoms of DM1 and thus their potential should be explored via rigorous efficacy studies in appropriate preclinical models.

Reference:

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I.
Brockhoff M, Rion N, Chojnowska K, Wiktorowicz T, Eickhorst C, Erne B, Frank S, Angelini C, Furling D, Rüegg MA, Sinnreich M, Castets P.
J Clin Invest. 2017 Jan 9. pii: 89616. doi: 10.1172/JCI89616. [Epub ahead of print

Biomarkers are a major interest for myotonic dystrophy (DM), but understanding of their utility (Context of Use) in clinical trials can be elusive. The ‘flavors’ of biomarkers relate to the ways they are utilized: diagnostic, prognostic, predictive, pharmacodynamics (PD) and pharmacokinetic (PK). The right biomarkers are invaluable in selecting/stratifying patients, determining on-target activity, and dosing and assessing efficacy and safety of candidate therapies. Arriving at the “right” biomarkers to minimize uncertainty and aid decision-making is essential, but nontrivial, as experiences in Duchenne have so clearly shown.

A simplistic view is that a molecular endpoint identified in a laboratory study can be a panacea in accelerating drug approval. The reality is that, in moving beyond the discovery phase, a range of questions, from methodological to interpretive, must be answered before a biomarker has validity.

Given the high bar for regulatory qualification, biomarker studies ultimately must reside within collaborative networks that recognize that no one gets to the solution alone. A key barrier to overcome is that biomarker qualification is uncharted territory for most academic researchers.

Two recent initiatives, the BEST Resource (Biomarkers, EndpointS, and other Tools) and the Framework for Defining Evidentiary Criteria for Biomarker Qualification, are aimed at clarifying terminology and process with all stakeholders, and thereby accelerating qualification and use of biomarkers in therapy development programs.

The FDA-NIH Joint Leadership Council, dedicated to improving regulatory science, developed the BEST Resource. BEST initially focused on harmonizing the terminology of translational science and biomedical project development. The intent is to provide clarity and consistency in communications among all stakeholders. The BEST glossary is an invaluable resource, going well beyond biomarker and endpoint terminology to provide a wealth of examples and informational links.

The Framework for Defining Evidentiary Criteria for Biomarker Qualification, a partnership led by the Foundation for the NIH that includes NIH, FDA, PhRMA, the Critical Path Institute and pharmaceutical companies, provides “a general framework to assist the development of biomarkers for qualification, to improve upon the quality of submissions to the FDA and to clarify the evidentiary criteria needed to support the biomarker’s "Context Of Use" (pdf). The ready availability of these criteria increases transparency of the qualification process and thereby facilitates interactions between biomarker developers and FDA.

What opportunities exist to exploit these new tools in biomarker development for DM? In a recent DM Research News, MDF highlighted the potential for FDA biomarker qualification of a panel of splicing events identified with the Myotonic Dystrophy Clinical Research Network (DMCRN). The recent clarification of evidentiary standards will markedly aid this effort. In addition, the Wyck Foundation and MDF recently funded Dr. Thurman Wheeler (Massachusetts General Hospital) to explore miRNAs in serum and urine as DM1 biomarkers. While this is a discovery-phase project, it’s important that the new qualification guidance is taken into account even by studies at such an early stage.

A recent publication by Ms. Alessandra Perfetti, Dr. Fabio Martelli and colleagues (IRCCS Policlinico San Donato) piloted circulating miRNAs as putative biomarkers for DM1. Dysregulated miRNAs included miR-1, miR-27b, miR-133a/-133b, miR-140-3p, miR-206, miR-454 and miR-574. Elevated miRNAs correlated with impaired muscle strength and elevated MCK, and could readily distinguish DM1 (103 subjects) from controls (111). Some of the miRNAs identified in DM1 patient samples are non-specific in that they also are dysregulated in Duchenne (miR-1, mIR-206 and miR-133a/-133b), but that does not preclude their potential value as prognostic, predictive, PD or PK biomarkers in DM1. Before a biomarker can be qualified, more extensive studies must assess how this miRNA profile links to pathogenic or regenerative processes across multiple organ systems, and show if these miRNAs are suitable in tracking disease progression and/or drug efficacy.

To achieve qualified DM biomarkers, we all must speak the same “BEST” language and assimilate, rather than silo, lessons learned from each study. But most of all, the DM research community must adopt a highly collaborative culture (valuing community needs over individual publications), since validated, quantitative assays, well-powered and phenotypically rich data sets and inter-site validation are essential in navigating the pathway to effective drug development tools.

Reference:

BEST (Biomarkers, EndpointS, and other Tools) Resource
FDA-NIH Biomarker Working Group.

Framework for Defining Evidentiary Criteria for Biomarker Qualification: Final Version. Evidentiary Criteria Writing Group

Validation of Plasma MicroRNAs as Biomarkers for Myotonic Dystrophy Type 1.
Perfetti A, Greco S, Cardani R, Fossati B, Cuomo G, Valaperta R, Ambrogi F, Cortese A, Botta A, Mignarri A, Santoro M, Gaetano C, Costa E, Dotti MT, Silvestri G, Massa R, Meola G, Martelli F.
Sci Rep. 2016 Dec 1;6:38174. doi: 10.1038/srep38174.