Model organisms have yielded important insights into neuromuscular diseases. Findings from the relatively straightforward models now link unstable expansions of CTG and CTTG repeats to the phenotypes of myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2) respectively. Yet, it would be a mistake to assume that we understand all therapeutically relevant pathogenic or disease modifying mechanisms in DM. A particularly vexing issue has been how DM1 and DM2 are mediated by MBNL sequestration, but yield phenotypes of differing severity. New fly models may provide some insights.

Novel Models for DM1 and DM2

To address divergent aspects of pathology in DM1 and DM2, Dr. Rubén Artero and colleagues (University of Valencia) generated and evaluated novel Drosophila models expressing the respective repeats (250 CTG or 1,100 CCTG) in skeletal and cardiac muscle. Flies expressing 20 CTG or CCTG repeats were also generated and used as controls.

Similar, Severe Phenotypes Seen in DM1 and DM2 Fly Models

The investigators showed that the established molecular features of DM—formation of nuclear aggregates, MBNL depletion, RNA splicing defects and upregulation of autophagy genes (Atg4, Atg7, Atg8a, Atg9 and Atg12)—occurred in their DM1 and DM2 models. They establish that expanded CCUG repeat RNA has similar potential in vivo toxicity as does CUG repeat RNA. Both models had severe skeletal (50% reduction in fiber cross-sectional area) and cardiac muscle phenotypes, and reduced survival. Cardiac dysfunction included altered systolic and diastolic intervals, deficits in contractility (percentage (%) of fractional shortening) and arrhythmias; some cardiac measures showed higher severity in the DM2 model fly. 

Do Unknown Factors Mitigate Cardiac Disease in DM2?

While understanding that no model organism can actually be said to “have DM,” fly and mouse models have informed understanding and treatment of DM. In the DM2 fly model, the cardiac phenotype is more severe than is seen in DM2 patients. The investigators suggest that while both CUG and CCUG expanded repeat RNA have the potential to cause severe striated muscle phenotypes, there may be mechanisms beyond the well-established toxic RNA pathway that reduce the toxicity they observed in the fly in human DM2. These findings and models may have relevance for identification of genetic modifiers or as validation screens for small molecule drug development.

Reference:

Expanded CCUG Repeat RNA Expression in Drosophila Heart and Muscle Trigger Myotonic Dystrophy Type 1-like Phenotypes and Activate Autophagocytosis Genes.
Cerro-Herreros E, Chakraborty M, Pérez-Alonso M, Artero R, Llamusí B.
Sci Rep. 2017 Jun 6;7(1):2843. doi: 10.1038/s41598-017-02829-3.

A recent review article makes the case that DM is a brain disease and that better understanding of and treatment strategies for the neurological consequences of DM are essential.

Considerable Gaps Exist in Understanding of the CNS in DM

The central nervous system (CNS) consequences are arguably among the least understood aspects of myotonic dystrophy (DM) and certainly have received only modest attention in drug development, yet:

• Grant applications—or at least successful ones—in this niche are few. The National Institutes of Health’s (NIH) categorical spending database reports that approximately $1.6 million of the $8.8 million awarded for DM in fiscal 2016 is for grants focused solely on the CNS (just one R01 and one P01 that is in its last year);
• Savvy industry researchers recognize the considerable contribution that CNS sequela make toward the overall burden of disease;
• Exploration of the CNS in has produced sparse natural history data and few insights into biomarkers that can provide early indications of target engagement and modulation and clinically meaningful endpoints.

A Status Report

Drs. Genevieve Gourdon (French National Institute of Health and Medical Research) and Giovanni Meola (University of Milan) have published a provocative review article addressing current status of understanding of CNS-related symptoms and the development of tools and therapeutic strategies to address them in DM.

An overall premise of this review, while acknowledging the considerable skeletal and cardiac muscle involvement, is that DM is a brain disease. The authors do acknowledge an essential barrier in moving forward toward CNS-targeted therapies—that the current level of understanding of how repeat expansion-triggered molecular changes link to CNS phenotypes in DM is only a shadow of the mechanism-to-phenotype understanding that we have for skeletal muscle.

The authors review current knowledge of the neuropsychological, cognitive and other CNS signs in CDM, DM1 and DM2, based on available functional and imaging methodology. A gap in longitudinal data, understanding how CNS symptoms progress with age, was identified as particularly acute. To drive interventional studies, it is necessary to establish strong correlations between CNS functioning and endpoints (such as imaging) that can be easily and reproducibly assessed in clinical trials of manageable duration. While we are not yet to this point, the authors note that small studies linking skeletal muscle and neurologic changes are suggestive of common disease mechanisms and highlight the potential that further data may support the linkage of muscle and CNS endpoints in the same clinical trial.

While the invasive splicing biomarkers developed for skeletal muscle-targeted drugs are not available for the CNS, assessment of proteins or exosomal RNA in blood or CSF may provide insights into patient neurological status. The authors’ literature review suggests that correlations between biomarkers and CNS functional status may be weak, at least for studies reported thus far. Instead they point to the value of cell and animal-based models as a means to develop sufficient scientific rationale to drive human clinical trials.

Finally, Drs. Gourdon and Meola give their assessment of putative strategies to therapeutically target the CNS in DM: the primary DNA mutation, toxic RNA, mediator proteins, or the variety of downstream targets arising from specific gene mis-splicing events. Nearly all of the data supporting these various strategies has been developed in studies of skeletal muscle, while conceptually applicable to the CNS, but will encounter both drug delivery and potentially different toxicity questions.

Taken together, the difficulties of assessing the CNS in DM, identifying meaningful endpoints for clinical trials, and ultimately establishing the effectiveness of CNS targeted therapies is captured in the author’s observation of the complex interrelationship of neurological, psychological and social factors in DM. Yet it is essential that we find ways to address the critically important neurological sequela of DM.

Steps to Address the Problem

The authors point to knowledge gaps—longitudinal natural history studies, linkage of imaging and other biomarkers to CNS phenotypes, and better-powered studies focused on more homogeneous cohorts—as important to the path forward. For its part, MDF has organized a forum, Bringing the Patient Voice to CNS-Targeting Drug Development in Myotonic Dystrophy, to bring in the invaluable patient perspective at the IDMC-11/MDF Annual Conference in San Francisco, on September 9, 2017.

Reference:

Myotonic Dystrophies: State of the Art of New Therapeutic Developments for the CNS
Gourdon G, Meola G.
Front Cell Neurosci. 2017 Apr 20;11:101. doi: 10.3389/fncel.2017.00101. eCollection 2017.

MDF staff recently attended the 2017 annual meeting of the American Academy of Neurology, in Boston, MA. Here are highlights from that meeting.

Clinical and histopathological findings in myotonic muscular dystrophy type 2 (DM2): retrospective review of 49 DNA-confirmed cases.
Bhaskar Roy, Qian Wu, Charles Whitaker, and Kevin Felice.

A better understanding of the natural history of DM2 is essential to the design of interventional clinical trials. This poster reviewed clinical profiles of a cohort of 49 confirmed DM2 cases seen over 24-years at Beth Israel. Proximal lower limb weakness was the most predominant symptom, although weakness ranged from absent to severe. Myotonia, grip strength, and FVC also showed considerable variation. Approximately half of study subjects had cataracts.

Evaluation of postural control and falls in individuals with myotonic dystrophy type 1.
Katy Eichinger, Jill R. Quinn, and Shree Pandya.

Clinical trial endpoints that measure parameters meaningful to patients will be necessary for registration trials in myotonic dystrophy (DM1). This poster presented an assessment of postural control and self-reported falls in a cohort of 34 DM1 subjects, studied over a 12-week observational period. Postural sway measurements in DM1 subjects differed significantly from norms and showed good test/re-test reliability. None of the postural measures used were predictive of fall status, although this may be due to the small sample. Further evaluation of postural status may yield reliable, clinically meaningful clinical trial endpoints.

Identification of dysregulated musclin expression and elevated atrial natriuretic peptide levels in adult and congenital myotonic dystrophy.
Donald McCorquodale, Katie Mayne, Brith Otterud, Diane Dunn, Bob Weiss, and Nicholas Johnson.

Understanding tissue-level molecular changes in DM can guide biomarker development as well as identify novel therapeutic targets. This poster addressed two components of a pathway that mediates response to exercise. Musclin expression, an upstream regulator of atrial natriuretic peptide (ANP), increased in skeletal muscle of congenital myotonic dystrophy (CDM) and DM1, accompanied by increases in ANP clearance receptor (NPR3) and ANP. Disregulated musclin/ANP signaling may be linked to weakness and exercise intolerance in CDM and DM1.

Correlation between MRI cerebral white matter changes, muscle structure and/or muscle function in myotonic dystrophy type 1 (DM1).
Cheryl Smith, Peggy Nopoulos, Richard Shields, Dan Thedens, and Laurie Gutmann.

Understanding any linkage between CNS and skeletal muscle changes in DM1 may provide insights into putative biomarkers and clinical trial endpoints. This poster presented pilot data on potential CNS contributions to skeletal muscle structure and function in a DM1 cohort. Data show correlations between an MRI measure (global cerebral fraction anisotrophy—a measure of white matter abnormalities) and both MRI measures of lower limb muscle structure and a lower extremity tracking task (a measure of functional weight bearing movement). The authors concluded that these data suggest that CNS changes in DM1 play a role in neuromuscular functional deficits.

Borderline CNBP CCTG expansions in myotonic dystrophy type 2 in over 16,000 specimens analyzed in a clinical laboratory.
Elise Nedzweckas, Rebecca Moore, Marc Meservey, Tara McNamara, Nicholas Tiebout, Zhenyuan Wang, Sat Dev Batish, and Joseph Higgins.

The frequency of DM2 expansions in the pre-mutation range (CCTG repeat length of approximately 177-372) is unknown. This poster from Quest Diagnostics utilized PCR, PCR repeat-primed, and Southerns to determine CNBP CCTG expansion lengths in 16,253 samples. The frequency of ‘borderline’ repeats was 0.97%, a value larger than in previously published studies. The potential for repeats in this borderline range to expand to pathologic lengths is, as yet, unknown.

Genetic markers of myotonic dystrophy type 1 (DM1) and Duchenne muscular dystrophy (DMD) in human urine.
Layal Antoury, Ningyan Hu, Leonora Balaj, Xandra Breakfield, and Thurman Wheeler.

Availability of a non-invasive biomarker to track target engagement/modulation of candidate therapeutics would be valuable to any DM clinical trial, and elimination of muscle biopsies would be critical for trials in pediatric CDM subjects. The platform presentation reported analyses of exosomal RNA in blood and urine of DMD, BMD, and DM subjects. Serum showed no differences between DM1 and controls. Several splicing event alterations known to change in skeletal muscle were not detected in urine. But, at least 10 transcripts were differentially spliced in urine that followed patterns seen in skeletal muscle and thus showed potential as non-invasive biomarkers. The source of differentially spliced transcripts in urine was thought to be the kidney or other urinary tract cells.  The group is working to correlate the pattern of splicing events detected in urine with phenotypic changes in DM1 patients.

Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 distal and proximal muscles.
Giovanni Meola, Laura Valentina Renna, Francesca Bose, Barbara Fossati, Elisa Brigozi, Michele Cavalli, and Rosana Cardani.

Metabolic dysfunction, including insulin resistance and increased risk of type 2 diabetes mellitus are characteristic of DM1 and DM2. While the insulin receptor (INSR) gene is known to be mis-spliced and links to the DM metabolic phenotype, other insulin signaling pathway components may be involved. This platform presentation presented data on insulin signaling pathway changes in DM1 and DM2 muscle biopsies. DM muscle biopsies showed increased fetal INSR isoform and altered expression and phosphorylation of selected proteins in the IR signaling pathway was seen in DM1 subjects. These effects were more pronounced in proximal versus distal muscles. The authors suggest that profiling of changes in INSR signaling pathways markers might emerge as a biomarker for clinical studies and trials in DM.

Increased EEG theta spectral power in polysomnography of myotonic dystrophy type 1 compared to matched controls.
Chad Ruoff, Joe Cheung, Jennifer Perez, Saranda Sakamuri, Emmanuel Mignot, John Day, and Jacinda Sampson.

Excessive daytime sleepiness and fatigue are hallmarks of DM—development of clinical endpoints to reliably evaluate these symptoms will help drive clinical trials. This poster presented data characterizing EEG spectra from nocturnal polysomnography in DM1 vs. controls. DM1 patients showed increases in wake after sleep onset and increased theta power in stage 2, stage 3, and all sleep stages combined when compared to control. EEG spectral power is being further evaluated as a putative biomarker.

Many of you have seen posts on social media about treatments they have received in other countries or heard about through friends, that include everything from dietary aids to gene therapy, and want to know how you can assess the possible benefits and risks of these "treatments." In this complicated therapy environment, how can patients make decisions about whether an available treatment or therapy is safe and effective? How can you tell the quacks from the cures?

In Pursuit of a Cure

MDF is committed to the pursuit of improved Care and a Cure for people living with myotonic dystrophy (DM). We’re a non-profit advocacy organization and there is no other reason for our existence. In pursuit of a cure, we fund research, support infrastructure projects for therapy development, recruit investigators to work on DM, educate drug regulatory agencies, and work with companies to help them see the opportunities and potential for investments in a new therapy for myotonic dystrophy.

Patients and their families know well that the search for a cure, or even a treatment that can mitigate symptoms of DM, is a long and arduous process. We have recently seen the development of IONIS-DMPK-2.5Rx ended because the oligonucleotide drug did not reach its target tissue (skeletal muscle) in concentrations adequate to have a meaningful effect. Fortunately, Ionis has reported that it has alternative compounds that appear to have better tissue-targeting and we hope to see these move toward clinical trials.

Sometimes it’s Complicated

More broadly, we have seen significant recent controversy and lack of agreement regarding therapies developed for other rare diseases, with insurance companies refusing to reimburse for drugs they claim do not have enough scientific evidence to demonstrate a clinically-meaningful effect for patients. We also know that the ‘placebo effect’ where patients report significant therapy benefit when actually on a placebo (a non-active substance with no therapeutic effect), can also complicate the discussion, particularly when a given therapy has a relatively small demonstrable impact. 

Stick with What Works

Oligonucleotide drugs can succeed as therapeutics. Biogen and Ionis collaborated on the development of Spinraza for spinal muscular atrophy. The two companies exercised considerable care in development of these drugs and sought FDA approval only after obtaining results from two international, placebo-controlled clinical trials. The key here is that considerable drug effect was demonstrated in a large cohort of patients enrolled in the clinical trials. As a consequence, the drug is now marketed for all types of spinal muscular atrophy and, while the cost of the drug is very high, many families are getting insurance coverage for Spinraza. The evidence had to be there for both therapy approval and insurance company reimbursement.

Achieving a drug that is proven to have a considerable level of effect on measures that are clinically-meaningful to DM patients is a central requirement for both drug approval and reimbursement. 

The therapies that we hope to achieve for DM will come only from this evidence-based drug development and approval process. MDF regularly meets with biotechnology and pharmaceutical companies—including ten companies in the last two months—providing information and making the case that DM represents a good investment with a clear pathway to drug approval. Any legitimate clinical trial will be listed in ClinicalTrials.gov, and information about legitimate DM studies and trials will always be circulated by MDF.

Dangers lie in the pursuit of quack “therapies.” A brief Google search will reveal fabulous claims of cures for just about any disease, if the patient will only travel to a developing country, with less regulatory oversight, for the ‘breakthrough’ therapy. Most often, the claims of effectiveness lack substantiation. These “therapies” have certainly not gone through any drug regulatory agency for approval, and often supportive data has not even been published in a reputable scientific or medical journal. They are, to put it bluntly, quack “therapies” that are potentially harmful because safety data is often not there.

To the safety point, even in the U.S., unproven “therapies” that bypass FDA regulations have caused harm. Three women were recently blinded in Florida after receiving stem cell “therapy” injections for macular degeneration.

Do Your Homework

So, to steal from an old saying, ya can’t tell the quacks from the cures without a scorecard. If a DM therapy sounds too good to be true, the people behind it are probably just after your money. The reliable scorecard here is the physician who is knowledgeable of DM. If your doctor or any other reputable physician with an understanding of DM won’t prescribe the treatment, you probably should not be taking it. 

MDF is also happy to help you understand whether something is in a legitimate clinical trial, an approved therapy…or not. Browse the resources and tools available on the MDF website or call the MDF Warmline at 415-800-7777.

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.

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.

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.

Building a Better Mouse

MDF has entered into a one-year, $90,000 partnership with Dr. Cat Lutz and Jackson Laboratory (Bar Harbor, ME) to develop a new mouse model of myotonic dystrophy type 1 (DM1).

The most commonly used DM1 mouse model, the HSALR mouse developed in Dr. Charles Thornton’s lab, has been an invaluable contributor to the understanding of pathogenic mechanisms in DM1 and has served in the development of preclinical rationale (proof of concept) to drive clinical trials in DM1. HSALR mice exhibit aberrant splicing of many genes that are mis-spliced in DM1, including Clcn1 and, consequently, show prominent myotonia. However, this model has limitations that include expression of the untranslated CUG repeat in an mRNA unrelated to DM1, tissue-limited CUG repeat expression (e.g., absent from critical brain and heart tissues, since regulation is driven by the HSA promoter), and the mouse lacks many of the multi-systemic features of DM1.

The deficiencies of the HSALR mouse model may be a consequence of the insertion site, length, developmental expression and/or flanking sequences of the CUG repeat. The partnership with Jackson Laboratory addresses these issues by seeking to develop a DM1 mouse model that more closely mimics the genetics of DM1. Dr. Lutz will develop a BAC transgenic mouse with insertion of an expanded repeat tract DMPK gene and flanking regions isolated from a DM patient BAC library. This new mouse should better replicate the molecular and cellular pathogenic mechanisms that operate in DM1, and may then better express the wider organ system involvement that is seen in DM1 patients.

By working with Jackson Laboratory, MDF intends to have the new DM1 model readily available to both academic researchers and drug developers at modest cost and without Intellectual Property restrictions within the next 12 months.

Creation and Distribution of DM Cell Lines for Research and Therapy Development

MDF is collaborating with the Human Cell and Data Repository (NHCDR), a joint venture involving the National Institute of Neurological Disorders and Stroke (NINDS) and RUCDR Infinite Biologics at Rutgers University. The partners in the collaboration are dedicated to the development of new fibroblast and iPSC lines, including isogenic iPSC lines for neurological disorders.

Over the next year, the collaboration will develop DM1 and DM2 fibroblast cell lines and at least four iPSC lines each from DM1 and DM2 patient cells. Through this collaboration, we will achieve unencumbered access and distribution of cell lines essential to mechanistic and drug discovery studies in academia and companies.

Availability of quality controlled, isogenic iPSC lines will mitigate, if not eliminate, an early stage barrier to entry of biotechnology and pharmaceutical companies into high-throughput screening programs for DM1 and DM2. Given the multi-system consequences of DM, availability of iPSC lines is of particular importance, as they provide the means to derive myoblast, cardiomyoblast, neuronal, or other cell types for use in studies of tissue-specific disease mechanisms and/or testing and optimizing specifically targeted candidate therapeutics.

When available, the DM patient-derived cell lines will be accessed through the NHCDR on-line catalog. The ensuing collaboration between MDF and academic and government partners will assure the availability of critical patient-derived resources at modest cost and without Intellectual Property restrictions that could hinder commercial drug development.