For Ami Mankodi, M.D., it was love at first sight. When she was in the fourth grade in Mumbai, India, she remembers seeing a picture of a brain in a book and knowing then that she wanted to be “a brain doctor,” not yet aware of the word “neurologist.”

"I looked at the organ, and I said, ‘Mommy, I want to become this doctor,’" said Dr. Mankodi. "Something struck, and there was no other option in my life."

Now a principal investigator at the National Institutes of Health’s (NIH) National Institute of Neurological Disorders and Stroke (NINDS) in Bethesda, Maryland, Dr. Mankodi has been involved in research that has helped shape a fundamental biologic and molecular understanding of myotonic dystrophy (DM).

Dr. Mankodi has participated in important advances in understanding critical questions about myotonic dystrophy, and these advances have pointed the way toward therapeutic approaches to treating the disease. But many questions remain unanswered about DM progression and how to best measure the severity and progress of a patient’s individual condition, questions she is working to answer today.

Finding Targets

Dr. Mankodi earned her medical degree from Grant Medical College in Mumbai, India, before performing post-doctoral work in the lab of Dr. Charles Thornton at the University of Rochester. After seven years in Dr. Thornton’s lab, she then completed a neurology residency at Johns Hopkins Hospital. The research she conducted with Dr. Thornton included the creation of a mouse model for myotonic dystrophy type 1 (DM1) and provided evidence that the disease was RNA-mediated. 

The genetic mutation driving myotonic dystrophy causes expression of RNA that contains expanded repeating code in the portion of the RNA not involved in the production of protein. The repeats are associated with both skeletal muscle degeneration and the diminished ability of the brain to communicate with muscles to relax after activity. One thing that Dr. Mankodi and her colleagues discovered was that an effect of these repeats was to reduce the number of chloride channels on the muscles. These channels are needed to receive electrical impulses that instruct muscles to relax and restore to a normal state after they have been constricted for activity. In simple terms, it is why someone who has myotonic dystrophy may find it difficult to open their hands after grasping an object, relax their jaw or tongue, or experience other muscle cramping symptoms of myotonia. 

The good news, according to Dr. Mankodi, is that it points the way to a therapeutic approach because it suggests researchers may be able to restore normal function with drugs designed to bypass errors in RNA, such as so-called antisense therapies that are in development today. 

“We didn’t even know 25 years ago where the gene defect was, and that was 100 years after the first clinical description,” Dr. Mankodi said. “In the last 25 years since gene discovery, we have come a long way to understanding the disease mechanism.”

Unanswered Questions

Despite advances that Dr. Mankodi and other researchers have made in the understanding of myotonic dystrophy, much remains unknown about the disease. A component of Dr. Mankodi’s research today is aimed at understanding how the disease progresses. Because there is wide variation in the severity of symptoms, the constellation of symptoms any one patient will develop, and the rate of progression of the disease, such an understanding is critical to improving treatments and developing therapies. A better understanding of the disease will help researchers establish meaningful endpoints to assess the effectiveness of potential therapies in clinical trials, and consistent ways to measure improvement or decline in those living with the disease. 

In 2011, MDF awarded funding to establish the first-ever Myotonic Dystrophy Clinical Research Network (DMCRN), research infrastructure co-led by Drs. Charles Thornton and Richard Moxley, III of the University of Rochester. The DMCRN was originally located at five academic institutions around the U.S. and was created in part to prepare standardized trial sites for potential therapeutics working their way toward human clinical trials. NIH is one of now eight medical centers participating in the network and Dr. Mankodi serves as a primary investigator. Her work there focuses on developing tools to measure the severity and progression of the disease. 

“We need to develop more tools and more community effort,” said Dr. Mankodi. “We are, as part of the clinical research network, trying to define the disease status, the disease burden, the disease progression and trying to identify reliable outcome measures that can be applied to therapeutic trials. Efforts are being made in this direction.”

As an example, Dr. Mankodi points to a recently-concluded study at six of the DMCRN sites to see how consistent measurements are in the same patient between three-month time points and between two sites. A new 500-patient study will launch this summer that will gather disease progression and other natural history information, as well as seek to identify genetic modifiers that scientists believe partially control the disease severity patients experience.

Dr. Mankodi is also working to develop tools to measure muscle strength and muscle relaxation time in the hands. At first, she and her team tried to do this with a glove but found it wasn’t a reliable approach because of different hand sizes. In a new tool, markers are placed on the hand and read by a computer using laser trackers. She said they have already developed such a device for the ankle. Dr. Mankodi and her team are also working to develop clinical and imaging biomarkers of pulmonary function. Through the DMCRN, they collected tissue and blood samples in one study to look at biomarkers over the course of time. More than 100 patients were enrolled in that study. 

But even with the unknowns, researchers are trying to decipher, Dr. Mankodi is optimistic about the potential of developing therapies to treat myotonic dystrophy. To get there, though, she believes collaboration will be critical. 

"We are still at very early stages, but the momentum is increasing and driving interest," she said. "It’s going to involve patients and patient support organizations like MDF, the [pharmaceutical] industry, researchers, and regulators. These are the key components, and we need to bring the pieces of the puzzle together. It’s community-wide action that will be needed, and that is exactly what’s forming the basis of the Myotonic Dystrophy Clinical Research Network. The steps are being taken."

Dr. Mankodi will speak at IDMC-11 in September 2017 at the upcoming biennial global conference of approximately 400 DM researchers. The International DM Consortium meeting brings together scientists, clinicians, associations and patients to accelerate clinical and fundamental myotonic dystrophy research. IDMC-11 will occur this year in conjunction with the 2017 MDF Annual Conference. Both events will be held in San Francisco, California.

Gene Editing for DM

Gene editing has garnered considerable publicity as the newest technology with potential for developing therapies for rare diseases. MDF previously published a primer, titled "Using Gene Editing to Correct DM," on the CRISPR/Cas9 technology that has been heavily promoted in the media.

Gene editing technology uses molecular mechanisms that were first developed in bacteria as a shield against invasion from viruses. This approach is rapidly moving into clinical trials for a select group of diseases—those where cells can be isolated from the body, edited, and then returned to patients as a viable treatment for the disease. These diseases are predominantly disorders of the blood and cancers, and several clinical trials are recruiting patients in China (HIV-infected subjects with hematological malignances; CD19+ refractory leukemia/lymphoma; esophageal cancer; metastatic non-small cell lung cancer; EBV-associated malignancies). At least one trial has been approved in the U.S. by the Food and Drug Administration (FDA) and is expected to start soon (this is also for a set of cancers).

For myotonic dystrophy (DM), multiple organ systems are affected and we cannot take the simple path of editing and returning cells to the body—treatment must address simply too much body tissue mass, including the brain, the heart, skeletal muscles, the gastrointestinal system, and other organs that are affected. Thus, for CRISPR/Cas9 to “work” in DM, the gene editing reagents will have to be efficiently delivered to virtually every cell in patients and effectively execute the deletion of CTG and CCTG repeat expansions from the DNA. The delivery of gene editing reagents into patients is an incredibly difficult undertaking and is likely years away from clinical trials in any disease.

Could a Modified CRISPR Technology be Effective in DM?

Investigators at the University of California San Diego, the University of Florida, and the National University of Singapore have recently reported early research that potentially ‘repurposes’ gene editing technology for a set of RNA disorders—myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), a subset of Lou Gehrig’s disease (ALS) patients and Huntington’s disease. They have modified the Cas9 enzyme so it is targeted to toxic RNA, instead of the expanded DNA repeats in these diseases.

The researchers have optimized Cas9 so that it can specifically target and degrade expanded repeat RNA for DMPK and CNBP genes. In many ways, this is similar to the approach that Ionis Pharma is using to target CUG repeats RNA in DM1. 

Their development of an RNA-targeted Cas9 results in the degradation of toxic RNA, an increase in the MBNL protein, and reduction or elimination of the gene splicing defect that characterizes DM. The strategy uses gene therapy vectors to delivery the modified Cas9 enzyme. If this approach were to be effective, it’s likely that patients would only need a single intravenous injection to treat skeletal muscles, the heart, and the gastrointestinal system; because gene therapy does not cross the blood brain barrier, a second injection may be needed, into the fluid around the spinal cord, to treat the brain. To work toward clinical development, the researchers have formed a biotechnology company to raise funding and move the candidate therapy forward.

We Still Have a Considerable Way to Go Before this Novel Strategy is in the Clinic

While this approach shows promise, we should be cautioned that studies thus far have only tried the new experimental therapy in patient cells in tissue culture. Therapy development has to pass through preclinical testing in appropriate mouse models, preclinical safety testing and approval by the FDA before the first clinical trial can be launched. Importantly, this effort represents yet another shot on goal to develop a novel therapeutic for DM1 and DM2. MDF monitors all drug development efforts and will keep the community informed as to their progress.

Gene Editing by CRISPR/Cas9 is Here, but for Very Specific Diseases

Removal of expanded CTG or CCTG repeats using CRISPR/Cas9 gene editing technology is being explored as a potential strategy for therapy development in DM (see prior DM Research News article "Gene Editing for DM"). A search of the ClinicalTrials.gov database indicates that gene-editing trials are now recruiting for some indications in China (HIV-infected subjects with hematological malignances; CD19+ refractory leukemia/lymphoma; esophageal cancer; metastatic non-small cell lung cancer; EBV-associated malignancies) and regulatory approval has been granted for at least one gene editing trial in the U.S. (for various cancers).

These first trials invariably involve editing cells that are easily isolated from patients, edited ex vivo, and then cells are restored, as this approach avoids the considerable technical difficulties and safety issues of delivering gene-editing reagents to in vivo targets. Indications, like DM, where gene editing must be done in vivo, have a more difficult path.

Steps Toward, and Beyond, Removing DM Expanded Repeats

Bé Wieringa and colleagues previously evaluated the feasibility of using CRISPR/Cas9 technology to remove long CTG repeat tracks from DMPK both ex vivo, in DM1 patient myoblasts, and in an animal model, HSALR mice. Their studies suggest that a dual cleavage strategy (cutting from both sides of an expanded CTG track) is necessary to minimize unpredictable genomic changes.

A new publication in Cell, by co-lead authors Ranjan Batra (an MDF fellow) and David Nelles and their colleagues, provides new insights into a potential redirection of gene editing technology as a candidate therapeutic for DM. Their development of an RNA-targeting Cas9 (RCas9) of a size compatible with AAV packaging and delivery, represents a novel strategy to use Cas9 to target not DMPK, or CNBP, but rather their expanded repeat RNA.

Batra, Nelles, and colleagues first developed a Cas9 devoid of nuclease activity (dCas9) and linked it to GFP, allowing them to localize and track RNA carrying CUG and CCUG expansions. This tool allowed them to optimize sgRNA design to specifically target toxic DMPK RNA, including that in nuclear foci. At higher doses of dCas9-GFP with the optimal guide sequence, they showed that binding to CUG and CCUG repeat RNAs resulted in their destabilization and elimination. Further structure-activity evaluations of the RCas9 resulted in constructs that cleave expanded CUG and CCUG repeat RNA and are compatible with an AAV-packaged therapeutic efficient at degrading toxic DMPK transcripts at low concentrations.

The research team then evaluated the efficacy of RCas9 in DM patient-derived myoblasts and myotubes—the approach proved effective in eliminating expanded repeat RNA, nuclear foci, and the splicopathy in DM1 and DM2 cells. Looking at one aspect of a putative therapeutics’ safety profile, they observed few unintended alterations to the transcriptome of myotubes exposed to RCas9 (these may be due to experimental environment, but further testing is essential if the approach is to move toward the clinic).

Targeting the RNA, not the Gene

The approach of using a modified Cas9, RCas9, which is targeted to expanded DMPK or CNBP RNA, represents a compelling new therapy development strategy for DM. This approach does not ‘correct’ the genome, as with traditional CRISPR/Cas9 strategies, but eliminates the toxic RNA in a manner similar to the antisense oligonucleotide therapies under development for DM1. While AAV delivery of RCas9 is required, a considerable hurdle, the RCas9 approach may overcome some of the barriers of targeting the expanded repeat track in the genome itself with CRISPR/Cas9. Ultimate head-to-head testing of RCas9 and antisense oligonucleotides may yield the optimal strategy for treating DM.

Reference:

Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9.
Batra R, Nelles DA, Pirie E, Blue SM, Marina RJ, Wang H, Chaim IA, Thomas JD, Zhang N, Nguyen V, Aigner S, Markmiller S, Xia G, Corbett KD, Swanson MS, Yeo GW.
Cell. 2017 Aug 10. doi: http://dx.doi.org/10.1016/j.cell.2017.07.010 [Epub ahead of print]

Understanding cardiac and other myotonic dystrophy (DM) risk factors and planning for the known complications of DM that may affect you someday can help protect and maintain your quality of life and that of your loved ones. Cardiac complications are the highest-priority care consideration for doctors treating patients with myotonic dystrophy type 1 (DM1) (as identified by expert clinicians in the forthcoming care guideline, "Consensus-based Care Recommendations for Adults with DM1"). As a result, researchers have been trying to understand the factors that may increase the risks of cardiac disease for DM patients.

Dr. Caroline Chong-Nguyen at the Sorbonne Paris Cité University and her colleagues recently published a study in which they looked at DM1 repeat length and its relationship to the risk of cardiac disease. This was a large study of the data in the French patient registry, which tracks patients' symptoms and information over time to understand disease progression and other important information. Eight hundred fifty-five patients with genetically-confirmed DM1 were followed for an average of 11.5 years in order to gain insight into how repeat length could predict cardiac events. Importantly, the research team considered many other factors (such as age, sex, and presence/absence of diabetes) to ensure that their data was not confounded by other variables.

The research team showed that death, sudden death and other adverse cardiac events were linked to DM1 repeat length. Heart rate was higher and conduction system disease was more prevalent in subjects with larger repeats. They found that each 500 repeat increase was associated with 1.5-fold higher risk of death from all causes. Patients with longer repeat lengths also were more likely to have a permanently-implanted pacemaker. 

These findings support taking a more aggressive approach toward screening DM patients for adverse cardiac events, particularly for DM1 patients at the higher end of the range of repeat lengths. Knowing your repeat will help you have discussions with your physician about monitoring and managing your level of risk for cardiac disease.

Recent studies suggest that the molecular basis of congenital myotonic dystrophy (CDM) differs from that of myotonic dystrophy (DM) type 1 (DM1). Epigenetic changes upstream of the DMPK locus appear to be a co-requirement, along with a threshold repeat expansion length, as a trigger for CDM. Yet, the basis for the considerable phenotypic differences between DM1 and CDM, downstream of genotypes, is poorly understood.

Understanding the divergence of the CDM and DM1 phenotypes may be found in the timing of the critical molecular events—while DM1 is driven by MBNL depletion and reversion to developmentally-regulated alternative splicing events, the severe phenotype of CDM may be linked to disruption of prenatal transitions in alternative splicing essential to normal muscle tissue development. However, little information has been available to support that hypothesis.

Thomas and colleagues (University of Florida and Osaka University Graduate School of Medicine) tested the hypothesis that prenatal depletion of MBNL and disruption of RNA alternative processing pathways critical to myogenesis (and likely other tissue-specific events) explains the severity of CDM. An MDF fellow, Łukasz Sznajder, contributed to this work.

These investigators utilized RNAseq to compare pre-mRNA processing in skeletal muscle biopsies of CDM, DM1, and individuals carrying DM1 pre-mutations. Their data show that alternative splicing events were highly conserved between DM1 and CDM, but consistently showed greater severity in CDM. Similarly, polyAseq identified a pattern of alternative polyadenylation in CDM samples that was similar to DM1, but also more severe.

Working from the model that in utero alternative splicing contributes to the severity of CDM, the team used existing RNAseq data sets to conduct in silico evaluations of RNA processing during in vitro differentiation of human primary myoblasts. They found that RNAs relevant to CDM showed prenatal isoform transitions that were predicted by the models of in utero consequences of expanded CUG repeats.

To extend their in silico findings, the investigators tested (a) the role MBNL plays in regulating RNA processing during myogenesis and (b) the linkage between RNA processing defects and CDM-like phenotypes using double (Mbnl1, Mbnl2) and triple MBNL (Mbnl1, Mbnl2, Mbnl3) knockout mice. In aggregate, these studies showed that double knockout mice developed a severe splicopathy and congenital myopathy, while data from the triple knockout suggests that Mbnl1 and Mbnl2 loss represents the primary cause of the spliceopathy, but the deletion of Mbnl3 is responsible for more subtle alterations in hundreds of additional splicing events. Both models also showed dramatic changes in gene expression profiles (particularly in stress-related pathways that have been linked to CDM), with, again, greater severity in the triple knockout. 

Taken together, these studies provide important insights into how molecular pathogeneic mechanisms may distinguish CDM and DM1, specifically that the breadth and timing of expanded CUG repeat toxicity and the resulting RNA processing defects contribute to the severity of CDM. Splicing changes in RNAs essential for the development of skeletal muscle were shown to be both MBNL-dependent and to occur in utero, and thus were linked to perturbations of myogenesis and the ensuing congenital myopathy. The novel mouse models developed here provide an important framework for future mechanistic studies to understand the divergence of CDM and DM1 phenotypes and to inform therapy development strategies.

This peer-reviewed research article was accompanied by an editorial by Drs. Jagannathan and Bradley, appearing in the same issue of the journal. This editorial is also referenced below.

References:

Disrupted prenatal RNA processing and myogenesis in congenital myotonic dystrophy.
Thomas JD, Sznajder ŁJ, Bardhi O, Aslam FN, Anastasiadis ZP, Scotti MM, Nishino I, Nakamori M, Wang ET, Swanson MS.
Genes Dev. 2017 Jul 11. doi: 10.1101/gad.300590.117. [Epub ahead of print]

Congenital myotonic dystrophy-an RNA-mediated disease across a developmental continuum.
Jagannathan S, Bradley RK.
Genes Dev. 2017 Jun 1;31(11):1067-1068. doi: 10.1101/gad.302893.117.

Accurate assessment of endpoints that are clinically meaningful to the patient is essential for the regulatory approval of a candidate therapeutic. Many of the endpoints used in clinical trials for myotonic dystrophy (DM) type 1 (DM1) thus far do not meet this requirement and thus do not represent adequate registration endpoints. Such registration endpoints are the holy grail for DM. Tools must be validated to assess the diverse factors that contribute to fatigue in order to develop clinical trial endpoints and effective therapies.

Baldanzi and colleagues (University of Pisa) have published an evaluation of several instruments in a cohort of 26 subjects with the genetic and clinical diagnosis of DM1 and proposed a paradigm to assess central and peripheral fatigue. They defined central fatigue as a decrement in voluntary muscle activation during exercise related to cognitive/behavioral function. By contrast, peripheral fatigue was characterized as the consequence of altered transmission at the neuromuscular junction or muscular dysfunction. The authors suggest a protocol for evaluation as an assessment of fatigue in DM1. 

Fatigue is an important contributor to patient-reported burden of disease in DM1. However, across neuromuscular diseases, there has been considerable debate around both defining and measuring fatigue.

Objectively, fatigue is defined as a decrease in power (work performed over time). Fatigue may arise from having to operate at or near one’s maximal motor functional capacity—diminishment of that capacity leads to early onset of fatigue. Yet, another important disease burden in DM2, pain, limits the performance of work and thus the measurement of fatigue is confounded as patients may not want to exert maximal effort due to the discomfort it causes. 

Since patients are able to detect even subtle changes in fatigue, patient reported outcome measures (PROMs) have potential as clinical trial endpoints. Fatigue can have both central and peripheral origins, and its dual origin may impact therapy development strategies. Thus, for a multi-systemic disease like DM, it is particularly important that we have tools to quantitatively evaluate fatigue regardless of origin and, optimally, gain some insights into peripheral and central contributions. A PROM scale such as the Modified Fatigue Impact Scale (MFIS) has three subscales (physical, cognitive, and psychosocial functioning) that may, in part, help understand fatigue. Another commonly used scale, the Multidimensional Assessment of Fatigue (MAF), is valuable in assessing four dimensions of fatigue—degree and severity, distress caused, timing and impact on activities of daily living.

Because fatigue is multifactorial, studies are needed to evaluate and validate measures of fatigue. The Myotonic Dystrophy Health Index (MDHI) is a PROM that has been incorporated into many recent clinical studies and trials and includes separate question banks that assess fatigue, sleep, and cognition. Its fatigue component is thought to focus on muscle fatigue, muscle endurance, and “tiredness” arising from muscle. The sleep and cognitive components have been linked to CNS-based fatigue, including motivation and concentration. 

The complex etiology of fatigue in DM1 makes it difficult for individual instruments to dissect peripheral and central components of fatigue. Given its key role in the burden of DM, it is critical that validated measures of fatigue be incorporated into natural history studies and clinical trials. 

Reference:

The proposal of a clinical protocol to assess central and peripheral fatigue in myotonic dystrophy type 1.
Baldanzi S, Ricci G, Bottari M, Chico L, Simoncini C, Siciliano G.
Arch Ital Biol. 2017 Jul 1;155(1-2):43-54. doi: 10.12871/000398292017125.

In January 2017, Ionis Pharmaceuticals reported results of their phase 1/2 clinical trial of DMPKRx in subjects with DM1. While the field gained considerable insights into the compound, clinical endpoints and future clinical trial design, DMPKRx did not achieve sufficient exposure in skeletal muscle to have the desired effect on RNA splicing. An examination of the totality of data behind DMPKRx can yield further insights as Ionis develops the next generation of antisense oligonucleotide drug candidate for clinical trials in myotonic dystrophy (DM).

Preclinical Evidence Supported Development of Ionis’ Constrained Ethyl-modified Oligonucleotide for DM1

A strong collaborative team in academia and Ionis Pharmaceuticals has recently published their preclinical animal efficacy studies of ISIS 486178, a compound of a similar class to the DMPK antisense oligonucleotide used in the DM1 clinical trial, ISIS-DMPKRx.

The therapeutic candidate molecule, ISIS 486178, was selected after extensive optimization of both oligonucleotide sequence and backbone chemistry, with over 3,000 compounds screened for suppression of DMPK. The study evaluated a battery of molecular and functional endpoints in: (a) myotonic dystrophy type 1 (DM1) and control cell lines and (b) DMSXL mice dosed subcutaneously with the selected compound, ISIS 486178.

The candidate therapeutic produced a 70% reduction in expanded CUG repeat RNA and nuclear MBNL-RNA foci in mouse skeletal muscle and 30% reduction in cardiac muscle. DMSXL muscle histology, forelimb muscle grip strength and body weight were also improved, with no overt safety signals (endpoints: survival, liver enzymes, CPK, creatinine and genome-wide profiling) noted in either mice or cultured myotubes. Changes were not noted in brain DMPK RNA levels, a finding expected with systemic dosing of oligonucleotides. Prior studies of DM1 are supportive of muscle maturational defects as a component of the pathologic mechanism—treatment with ISIS 486178 largely restored the myofiber maturational profile in the soleus of DMSXL mice. DM1-related splicopathy is mild and variable in DMSXL, so drug effect on mis-splicing was not evaluated.

Preclinical Proof of Concept Achieved for Targeting Expanded DMPK RNA

Taken together, treatment of DMSXL mice with ISIS 486178 produced substantial and reproducible reduction in mutant DMPK transcripts, as well as phenotypic improvements. The constrained ethyl backbone chemistry used in ISIS 486178 exhibited differential exposure to two important DM1 targets, skeletal muscle (70% reduction in DMPK transcripts) versus heart (30%). Using their earlier generation oligonucleotide chemistry (2'-O-methoxyethyl modified or MOE), Ionis successfully partnered with Biogen to achieve sufficient CNS exposure after intrathecal delivery, ultimately leading to regulatory approval of Spinraza for all types of spinal muscular atrophy in late 2016. It appears that improving delivery of a DMPK-targeted antisense oligonucleotide is a viable path forward for DM1.

Next Steps

Data published by this investigative team provide a strong scientific rationale for targeting mutant DMPK with oligonucleotides operating by an RNase H mechanism. MDF has a BAC transgenic model under development at Jackson Laboratories that may be a better model for assessing efficacy in restoring splicing in the context of the DMPK locus, as well as assessing multi-system phenotypes. Finally, Ionis has publically announced an ongoing preclinical development program to obtain an antisense oligonucleotide with better exposure and intends to return to clinical trials in DM1.

Reference:

Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.
Jauvin D, Chrétien J, Pandey SK, Martineau L, Revillod L, Bassez G, Lachon A, McLeod AR, Gourdon G, Wheeler TM, Thornton CA, Bennett CF, Puymirat J.
Mol Ther Nucleic Acids. 2017 Jun 16;7:465-474. doi: 10.1016/j.omtn.2017.05.007. Epub 2017 May 17.

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.

DMPK CTG expansion length generally correlates with the severity of myotonic dystrophy type 1 (DM1), but is not fully prognostic of disease onset, course and severity. For congenital myotonic dystrophy (CDM), the apparent requirement for an epigenetic change upstream of the DMPK locus is apparently a co-requirement, along with a long CTG repeat. Moreover, the relationship between repeat expansion length and the cardiac phenotype in DM is a gap in our understanding of cardiac disease in DM1.

Multivariate Analysis of a Large Genetically Confirmed DM1 Cohort

Dr. Caroline Chong-Nguyen (Sorbonne Paris Cité University) and colleagues characterized the relationship between DMPK repeat expansion length and cardiac disease in a retrospective study of a cohort of 855 adult subjects from the DM1-Heart Registry. Subjects entered into the study had genetic analysis (Southern blot of peripheral blood) done at the time of their baseline cardiac investigations.

Genotyped patients were followed for a median of 11.5 years. The authors utilized a multivariate analysis that considered potential confounding factors, including age, sex, and diabetes mellitus.

Repeat Length is a Key Factor in Prognosis Even When Confounding Variables are Taken into Account

Survival of DM1 subjects was correlated with the quartile of CTG expansion size—37% mortality was reported in subjects with greater than 830 repeats. Across the range of repeat lengths studied, each 500 repeat increase was associated with 1.5-fold higher risk of death from all causes. Heart rate was higher and conduction system disease, left bundle branch block, and longer PR and QRS intervals were more prevalent in subjects with larger repeats. CTG length also associated with the presence of a permanently implanted pacemaker. Availability of extensive longitudinal data allowed the authors to report Kaplan–Meier estimates for survival, supraventricular arrhythmias, pacemaker implantation and sudden death.

This longitudinal study of a large cohort genotyped at the time of initial cardiac evaluation provides new insights into genotype-cardiac phenotype relationships in DM1. Overall, the authors showed that longer DMPK repeat expansions were correlated with the severity of cardiac involvement, including development of conduction defects, left ventricular dysfunction, supraventricular arrhythmias, the requirement for permanent pacing, sudden death and mortality. These findings support a more aggressive approach toward cardiac screening based on DMPK repeat length—the authors argue that care should be based on assessment of conduction system defects and other cardiac manifestations.

This peer-reviewed research article was accompanied by an editorial by Dr. Matthew Wheeler (Stanford University) in the same issue of the journal. This editorial is also referenced below.

References:

Association Between Mutation Size and Cardiac Involvement in Myotonic Dystrophy Type 1: An Analysis of the DM1-Heart Registry.
Chong-Nguyen C, Wahbi K, Algalarrondo V, Bécane HM, Radvanyi-Hoffman H, Arnaud P, Furling D, Lazarus A, Bassez G, Béhin A, Fayssoil A, Laforêt P, Stojkovic T, Eymard B, Duboc D.
Circ Cardiovasc Genet. 2017 Jun;10(3). pii: e001526. doi: 10.1161/CIRCGENETICS.116.001526.

Repeats and Survival in Myotonic Dystrophy Type 1.
Wheeler MT.
Circ Cardiovasc Genet. 2017 Jun;10(3). pii: e001783. doi: 10.1161/CIRCGENETICS.117.001783

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.