In November, MDF staff met with the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and the National Institute of Neurological Disorders and Stroke (NINDS) senior leadership and program/policy staff to discuss research opportunities and federal support for myotonic dystrophy (DM). Discussions focused on two areas: the scientific workforce and biomarker and registration endpoint development.

MDF reviewed efforts to support the scientific workforce through the MDF Postdoctoral Research Fellowship program and expressed support for NIAMS and NINDS efforts to extend their R01 paylines for new investigators. Dr. Steve Katz, Director, NIAMS, noted that 58% of K award recipients successfully transition to R type awards and encouraged clinical researchers in DM to utilize the K award mechanism. Dr. Walter Koroshetz, Director, NINDS, noted that 22 academic medical centers currently held NINDS R25 awards to support resident’s research in neurology; he encouraged faculty working on DM at these medical centers to take full advantage of the R25 awards.

MDF also encouraged NIAMS and NINDS to consider mechanisms to support young faculty seeking to renew their initial R01 awards. This is often a critical stage for young investigators (and NIH data bear that out), as they have had to set up their lab, produce on proposed projects, and gather preliminary data for specific aims of the renewal within the initial five or fewer years of funding. The NIAMS and NINDS directors indicated that they were sensitive to the issue. Dr. Katz noted the NIAMS STAR Program (Supplements to Advance Research from Projects to Programs). This program provides up to $150,000 per award in administrative supplements to young faculty on their initial R01 who need additional time and data to obtain either a renewal or a new R01 award. Young faculty with an initial R01 from NIAMS should review the program announcement.

MDF staff also reviewed the Foundation’s role in addressing opportunities and challenges along the entire therapeutic development pipeline. The potential for development and qualification of biomarkers for use in early phase clinical development was highlighted as a timely opportunity in DM. Likewise, overcoming the challenges in developing registration endpoints for drugs and biologics under development for DM were identified as a critical need.

NIAMS Staff pointed to RFA-AR-17-009, Research Innovations for Scientific Knowledge (RISK) for Musculoskeletal Diseases (R61/R33), a program designed for high-risk projects, as a potential means of funding biomarkers and clinical endpoints in DM. NINDS has a continuing program targeted at clinical trial readiness (including biomarker and endpoint development): PAR-16-020, Clinical Trial Readiness for Rare Neurological and Neuromuscular Diseases (U01).

Since the NIAMS initiative has only one application date, and the NINDS initiative is intended for mature projects, such as biomarker qualification efforts, MDF staff strongly encouraged both institutes to consider an initiative to facilitate early discovery and development of biomarkers and endpoint measures for DM. MDF believes the existing funding opportunities are important, and should be considered by investigators, but noted that early stage discovery projects in these areas may not compete well with hypothesis-driven applications and that a targeted initiative is a critical need for the DM field.

Taken together, MDF will remain engaged with the NIH, meeting on a regular basis to ensure that opportunities and critical needs in the DM field receive attention. DM researchers are encouraged to make opportunities and needs known to MDF staff so they can be incorporated into discussions with NIH leadership and program staff.

The multi-system involvement and heterogeneity that characterize myotonic dystrophy (DM) have fostered several efforts to design patient-reported outcome measures (PROMs) for clinical studies and trials. The intent of PROMs is to use patient feedback in design and implementation of validated questionnaires that can simultaneously capture changes across the challenging symptomology of DM while obtaining clinically meaningful information to support regulatory approval. While PROMs can prove insightful as an analytic tool for complex disorders, the potential barriers to PROM design, development, and interpretation are such that the Food and Drug Administration (FDA) developed a Guidance for Industry document (pdf) to aid in development of PROMs. Choice of PROMs for use in interventional trials must then be carefully informed.

Dr. Tara Symonds and her colleagues at Clinical Outcomes Solutions (COS) recently reported out a literature review of available PROMs that focus on type 1 myotonic dystrophy (DM1). COS a is health economics and outcomes research consulting group with considerable experience in understanding PROM design and implementation in clinical studies. Disclosure: The COS project was funded by Biogen, a company engaged in therapy development in DM1.

Dr. Symonds and her group evaluated a health status measure (MDHI), three activities of daily living scales (DM1-Activ, DM-Activc, and Life-H), two health related quality of life measures (INQoL & INQoL Serbian), and five sleep and fatigue measures (ESS, DSS, CFS, FSS, and FDSS) comparing their validity, reliability, and ability to detect change of each to guide choice of PROMs for use in DM1 studies.

MDHI was viewed as the only measure that attempted to capture all aspects of a DM1 patient's life that were impacted. Design of MDHI specifically for DM, internal consistency of the tool across domains assessed, test-retest reliability, and design in compliance with FDA’s Guidance for Industry were viewed as favorable traits. It was also noted that construct validity had been established for MDHI via comparison with a variety of existing functional measures (e.g., MMT, grip testing, and timed function tests).

DM1-Activ also was considered to have good validity and reliability and showed construct validity when compared to various manual testing measures and the Muscular Impairment Rating Scale (MIRS).

Most other PROMs assessed by the authors were viewed as more limited in capability and performance, and all PROMs were thought to require further assessment of responsiveness and meaningful change thresholds in interventional clinical trials. Dr. Symonds and team concluded that MDHI is arguably the best measure for use in clinical studies and trials provided that the critical areas of responsiveness and definition of meaningful change to patient are addressed. DM1-Activ also was deemed to have potential as a PROM in interventional trials, as long as content validity is explored further and the issues of responsiveness and meaningful change prove acceptable. Other measures were considered acceptable in evaluation of specific domains of the symptomatology of DM1.

FDA’s Guidance for Industry supports the use of well-designed and implemented PROMs as putative primary endpoint measures for clinical trials. Even as a secondary measure, a carefully selected PROM can bring considerable value to clinical trials, not the least is insight into meaningful benefit to the patient. The publication by Dr. Symonds and colleagues provides an evaluation of currently available tools by experts outside of the DM community. While current drug discovery and development efforts have focused on skeletal muscle function, desired treatments for DM will have to address a much wider disease burden. Validated and reliable PROMs with an ability to capture changes in multiple symptoms important to DM1 patients may prove to be a valuable tool in natural history studies and definitive clinical trials.

Reference:

A Review of Patient Reported Outcome Measures for Use in DM1 Patients
Symonds T, Randall JA, Campbell P.
Muscle Nerve. 2016 Nov 11. doi: 10.1002/mus.25469.

To date, technological hurdles have been a barrier to creating a mouse model for type 2 myotonic dystrophy (DM2), hindering understanding and treatment development for this disorder. But that’s about to change, thanks in part to the work of Łukasz Sznajder, Ph.D., a recipient of a 2016-2017 research fellowship grant from MDF.

"Currently, there are only cellular and fruit fly models available," says Dr. Sznajder, "and they’re not sufficient to understand the complex nature of DM2. A mouse model is urgently necessary to break this barrier."

Without mouse models it would have been impossible to develop the drugs now being tested to treat most neuromuscular disorders, including type 1 myotonic dystrophy (DM1).

A DM2 mouse model “will provide an excellent platform to evaluate DM2 phenotypes,” Dr. Sznajder says.

It would also allow researchers to study tissues that are hard to obtain from patients, such as those from the cardiac muscle and brain, and it would shed light on the differences between DM1 and DM2.

"My proposed model represents a unique opportunity to distinguish the differences between DM1 and DM2," Sznajder says. "We expect to answer puzzling questions, such as why there is no congenital-onset form of DM2," which is caused by several thousand CCTG repeats in the first intron of the CNBP gene.

"It is worth mentioning that the size of this mutation is several times that of the expansion that leads to DM1," Sznajder says. That, he notes, poses some challenges in developing a DM2 mouse model. "First, the amplification of even a few hundred repeats is not possible using conventional strategies. Second, the precise insertion of these repeats into the mouse CNBP gene is a highly inefficient process," he says.

"New technologies have remarkably improved the efficiency of genome engineering, and we hope to use these technologies to overcome the current challenges in DM2 modeling," he says.

Moving Toward DM2 Therapies

A mouse model will also allow Dr. Sznajder and his team to test therapeutic strategies for DM2 like antisense oligonucleotides and small molecules.

The antisense oligonucleotide-based drug IONIS-DMPKRx, designed to block harmful interactions between expanded RNA repeats and cellular proteins in DM1, is now in a phase 1-2 trial. Dr. Sznajder and his colleagues hope to develop a similar molecule to treat DM2.

"It is scientifically possible to adjust the oligonucleotide sequence to make it useful for DM2," Sznajder says. "However, a good mouse model of the disease is needed to test the efficiency of this or other approaches." His new DM2 mouse is expected to provide this vital tool.

A Passion for DM Research

Dr. Sznajder recently moved from his native Poland to realize his dream of working with a renowned DM researcher in the United States.

Coming of age in Poland in the 2000s, Sznajder decided to become a biomedical scientist, ultimately earning his doctorate in biotechnology and molecular biology from the Adam Mickiewicz University in Poznań in December 2015.

"For some time," he says, "I had dreamed about working at a prestigious university in the United States under the supervision of someone who would enable me to develop my scientific career while following my passion for research in myotonic dystrophy."

During his graduate studies, Sznajder was fortunate enough to work under Dr. Krzysztof Sobczak, who had been a postdoc in the laboratory of Dr. Charles Thornton, a DM researcher at the University of Rochester in New York state and a colleague of Dr. Maurice Swanson.

Under the mentorship of Dr. Sobczak, Sznajder started working on RNA toxicity and MBNL proteins, which he describes as "critical players in the molecular cascade of DM."

Dr. Sznajder is continuing his vital DM research under Dr. Maurice Swanson at the University of Florida.

Patient compliance with prescribed medicine has been the subject of many studies in common and rare diseases—as many as 50% of patients with chronic diseases do not follow the directions provided by their physicians or pharmacists regarding their medications. Compliance with instructions for prescribed medicines does lead to better health outcomes. Unfortunately, only limited information is currently available regarding which medications myotonic dystrophy (DM) patients take, and there is little understanding of barriers that may prevent better DM patients from complying more diligently with the prescriptions provided by their doctors.

In a recent study by Dr. Richard Moxley, III, MD, and colleagues at the University of Rochester, researchers assessed disease manifestations and adherence to medications for DM1 and DM2 patients. The study, titled “Medication adherence in patients with myotonic dystrophy and facioscapulohumeral muscular dystrophy,” was motivated in part by the fact that DM patients need to take multiple prescriptions to manage disease symptoms associated with a number of different body systems. The study also sought to understand how difficulty in swallowing, limited mobility, and reduced employment may impact DM and facioscapulohumeral muscular dystrophy (FSHD) patient compliance with prescribed medicines.

The researchers surveyed adult DM1 and DM2 patients enrolled in the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members at the University of Rochester. For both DM1 and DM2, muscle weakness was the symptom that patients most commonly viewed as an unmet treatment need, and for which they wanted new therapies to be developed. Patients also cited the need for treatments to improve mobility and reduce fatigue as high-priority issues in DM1, while DM2 patients reported pain as an unmet need. Many patients surveyed took six or more medications. Improved access to physical therapy, exercise, and mobility devices may help reduce reliance on some medications.

Most DM patients reported a good understanding of both their disease and the reasons that they were taking specific medications. Most participants in the study (93% of DM1, 88% of DM2 patients) reported that cost/insurance coverage was not a barrier to compliance with medications prescribed for their DM symptoms. Side effects of one or more medications were important compliance factors for a significant number of DM patients (35% in DM1, 49% in DM2), and were a factor that led to many patients discontinuing a medication (37% in DM1, 60% in DM2). Patients also identified difficulty in swallowing medicines in tablet or capsule form as a barrier to compliance with prescribed medication (33% in DM1, 21% in DM2).

Dr. Moxley and colleagues concluded that the symptoms of DM did not significantly impair patient adherence to medications prescribed for their multi-system disease. DM1 patients identified more of a need for new medications to manage their disease symptoms than patients with DM2. DM patient compliance with medications was, overall, better than literature reports for other chronic diseases, and participants in the study felt that their medications did not negatively impact their social or work lives. Finally, the authors of the study noted that community pharmacists can be an excellent source of advice when taking multiple medications and in overcoming barriers to compliance. Going forward, there is a need to identity and evaluate the effectiveness of specific medications that are used in DM, and to identify strategies to address barriers such as difficulty in swallowing, to help ensure optimal care for patients living with DM.

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.