GSK3β as a Drug Development Target for DM1

Published on Sun, 01/07/2018

Clinical Trials Targeting GSK3β

AMO Pharma is in clinical development with AMO-2 (tideglusib), an inhibitor of GSK3β signaling, for congenital myotonic dystrophy (CDM). A single blind, phase 2 trial has been initiated at Newcastle University to evaluate drug safety and efficacy in adolescent and adult patients with congenital and juvenile-onset DM1 (see

Studies from Dr. Lubov Timchenko and colleagues provided the initial preclinical rationale for the GSK3β target in a mouse model of DM1 (Jones et al., 2012). AMO’s clinical development of the GSK3β inhibitor, tideglusib, was, in part, based on these preclinical data. The GSK3β—cyclin D3—CUGBP1 pathway is of particular interest for CDM because of the potential role CUGBP1 plays in myogenesis. Now, additional studies have been undertaken to validate this novel target for drug development in CDM.

Evaluation of a GSK3β Inhibitor in Young HSALR Mice

Dr. Timchenko and colleagues at Cincinnati Children’s Hospital, Baylor College of Medicine, Centre Hospitalier–Université Laval Research Center, and Mount Sinai Hospital (Toronto) conducted evaluations of the validity of the GSK3β inhibition strategy in young (1.5 month old) HSALR mice. Two GSK3β inhibitors (6-bromoindirubin-39-oxime (BIO) and indirubin) were administered to mice, ip every 48 hours for 6 weeks. The group reported that early correction of GSK3β signaling was important for differentiation and long-term integrity of HSALR mouse skeletal muscle. Their data showed that correction of GSK3β—cyclin D3—CUGBP1 pathway activity was instrumental in the structural and functional changes in skeletal muscle. Using a Celf1 knockout model, the research team further established the connection between CUGBP1 levels and skeletal muscle development.

Relevance for Clinical Trials with Tideglusib

This latest study focuses on the validity of the GSK3β target for DM, as measured by changes in muscle histopathology and functional grip strength. These preclinical data show that the GSK3β target may be capable of modulating the progression and severity of the disease. However, differences in the drug employed and delivery regimen in this mouse study mean that it did not evaluate the efficacy of the tideglusib drug as a candidate therapeutic for DM. Rigorously-designed studies of tideglusib will be important to support the case for DM1 therapy development.


Correction of GSK3β at young age prevents muscle pathology in mice with myotonic dystrophy type 1.
Wei C, Stock L, Valanejad L, Zalewski ZA, Karns R, Puymirat J, Nelson D, Witte D, Woodgett J, Timchenko NA, Timchenko L.
FASEB J. 2017 Dec 4. pii: fj.201700700R. doi: 10.1096/fj.201700700R. [Epub ahead of print]

GSK3β mediates muscle pathology in myotonic dystrophy.
Jones K, Wei C, Iakova P, Bugiardini E, Schneider-Gold C, Meola G, Woodgett J, Killian J, Timchenko NA, Timchenko LT.
J Clin Invest. 2012 Dec;122(12):4461-72. doi: 10.1172/JCI64081. Epub 2012 Nov 19.

A Tool for Evaluating Drugs Targeting MBNL

Published on Sun, 01/07/2018

The Genesis of Drug Screening Assays

Early stage drug development programs are often predicated on the development of biochemical or cell-based assays that allow identification of candidate therapeutics that engage and modulate targets deemed to be potentially disease mitigating. To be efficient and effective such screening assays must meet accepted pharmaceutical industry criteria—guidance in development of acceptable assays has been provided as part of a preclinical research toolbox by the National Center for Advancing Translational Sciences (

Screening assays frequently arise from academic groups focused on both mechanistic and translational goals. While not typically in compliance with industry standards for high throughput screening of libraries that can exceed 1M compounds, such assays may provide important first steps in that direction.

A Novel Effort Towards a DM1 Drug Screening Assay

Dr. Krzysztof Sobczak and colleagues at Adam Mickiewicz University have developed a minigene-based assay to assess critical RNA binding protein sites/splicing regulatory regions in pre-mRNA that may have implications as a drug screening assay for DM1. Dr. Łukasz Sznajder, currently an MDF fellow at the University of Florida, was part of the team.

The research team first evaluated functional protein (MBNL)/RNA interactions, taking into account RNA primary and secondary structure in a design that allowed assessment by CLIP-seq or other means of transcriptome analysis. In an evaluation of the potential to assess splicing regulation, antisense oligonucleotides on two backbone chemistries (2’OMe and LNA) targeted to MBNL binding regions of Atp2a1 were able to inhibit exon 22 inclusion in the Atp2a1 transcript. The approach was validated using other transcripts regulated by MBNL.

They also designed hybrid Atp2a1 mini genes containing functional MBNL-binding motifs in introns and exons. These were used to establish that inclusion or exclusion of the MBNL motifs had the predicted effect on exon 22 splicing; results were confirmed in transfected HeLa cells. Taken together, they showed that MBNL-binding regulatory regions could be transferred from their original genetic context into a different mini gene transcript and still regulate alternative splicing.

In an initial proof of concept, the research team showed that the Atp2a1 mini gene could feasibly discern the potency of various interventions to disrupt MBNL binding. Inclusion of a reporter gene in the minigene construct may provide an effective assay for drug discovery via high-throughput screening.


Hybrid splicing minigene and antisense oligonucleotides as efficient tools to determine functional protein/RNA interactions.
Cywoniuk P, Taylor K, Sznajder ŁJ, Sobczak K.
Sci Rep. 2017 Dec 14;7(1):17587. doi: 10.1038/s41598-017-17816-x


The Origin of Insulin Resistance in DM

Published on Mon, 10/23/2017

Insulin Resistance and Diabetes in DM are the Product of INSR Mis-Splicing, Right?

Insulin resistance, impaired glucose utilization by multiple tissues (particularly in skeletal muscle), and multi-systemic consequences of type 2 diabetes mellitus represent impactful metabolic alterations that contribute to morbidity and mortality in DM1 and DM2. Understanding the molecular mechanisms behind insulin resistance will foster better treatments for patients living with DM. It is easy to conclude that we already understand the basis of insulin resistance in DM1—that it is a direct consequence of an already well-established mis-splicing and predominance of the fetal insulin receptor (INSR) transcript. Yet, that assumption has not been directly tested.

The Real Story Appears to be More Complex

An MDF Fellow, Laura Renna, and her colleagues in Milan have recently published a study that provides new insights into the pathogenesis of insulin resistance and diabetes in DM1 and DM2. This research team took the novel approach of evaluating INSR transcript and protein, and the status of downstream insulin signaling pathway components, in DM patient muscle biopsies and myotubes differentiated ex vivo in order to better understand the molecular causes of the metabolic phenotype that characterizes DM.

Despite observation of mis-spliced INSR in all DM1 (65% fetal isoform) and DM2 (50% fetal isoform) muscle biopsies, levels of INSR protein were not reduced when compared with controls. However, basal phosphorylation levels of Akt/PKB, p70S6K, GSK3β and ERK1/2 were altered, indicating potential compromise of signaling pathways downstream of the INSR. In keeping with DM1 pathophysiology, distal muscles (tibialis anterior) exhibited greater signaling pathway impairment than proximal (biceps brachii). 

To facilitate studies aimed at a better understanding of events downstream of INSR protein, the research team utilized DM patient-derived myotubes. In patient myotubes, no differences in INSR protein levels were detected between DM1, DM2, and controls.  But, analyses of glucose uptake showed reduced insulin-mediated stimulation in DM myotubes. Moreover, activation of both IRS1-Akt/PKB and Ras ERK pathways was impaired. Thus, deficits in insulin signaling in DM may not be the sole consequence of INSR mis-splicing, but rather may be due to dysfunction in downstream signaling pathways.

Taken together, the cellular/molecular mechanisms underlying reduced insulin sensitivity in DM may be more complex that has been appreciated. The Milan research team assessed insulin signaling pathways and concluded that perturbations of post-INSR signaling may well be a key factor in development of insulin resistance in DM, irrespective of any changes in INSR transcript splicing. The pathophysiological mechanisms underlying these alterations in post-receptor signaling proteins are currently unknown.


Receptor and post-receptor abnormalities contribute to insulin resistance in myotonic dystrophy type 1 and type 2 skeletal muscle.
Renna LV, Bosè F, Iachettini S, Fossati B, Saraceno L, Milani V, Colombo R, Meola G, Cardani R.
PLoS One. 2017 Sep 15;12(9):e0184987. doi: 10.1371/journal.pone.0184987.

RAN Translation in DM2

Published on Mon, 10/23/2017

Control of RNA Gain-of-Function Versus Toxic RAN Protein Mechanisms

Since the discovery of Repeat-Associated Non-AUG (RAN) translation, the pathogenesis of any disorder that is the consequence of microsatellite expansions must consider two distinct mechanisms—RNA gain-of-function (i.e., toxicity of expanded repeat RNA), toxicity of RAN proteins, or a combination of both mechanisms. For myotonic dystrophy (DM), the toxic RNA mechanism has predominated thus far, as there are only limited reports of the detection of RAN proteins in affected tissues. How these two potential pathogenic pathways in DM are regulated, and whether they may be in any way co-regulated, are currently open questions.

Muscleblind (MBNL) Plays a Key Regulatory Role Over RAN Translation

Dr. Laura Ranum (University of Florida) and colleagues have recently published studies that provide new insights into the regulation of molecular pathogenic pathways in DM2. Through assessments of expanded sense (CCTG) and antisense (CAGG) transcripts in DM2 autopsy brains and in vitro, they demonstrated the occurrence of bidirectional transcription. The research team also showed that transcripts containing a threshold repeat length produce the corresponding poly(LPAC) and poly(QAGR) RAN proteins, with protein production positively correlated with repeat length.  Cytoplasmic poly(LPAC) protein was localized to gray matter (neurons and glia), while poly(QAGR) was found in white matter, primarily in oligodendrocyte nuclei (but also in pathologic regions containing activated microglia). Using in vitro expression studies, the research team showed that both RAN proteins are toxic in neurons by mechanisms unrelated to RNA gain-of-function.

The production of poly(LPAC) protein was reduced or blocked by nuclear foci formation and retention of CCUG expansion RNA by MBNL1 binding. By contrast, CAGG transcripts do not bind MBNL1 and form nuclear foci, but rather are translocated to the cytoplasm, resulting in elevated poly(QAGR) RAN protein (CLIP experiments showed that poly(QAGR) protein does bind hnRNP A1).

A Model for Regulation of RAN Translation in DM2

Taken together, nuclear MBNL1 levels control the relative degree to which mis-splicing and RAN translation products contribute toward the pathogenesis of DM2. If free MBNL1 levels remain sufficient, expanded CCUG transcripts are retained in the nucleus, CCUG-mediated RAN translation is blocked, and disruption of splicing may not reach a level where functional consequences are observed. Depletion of MBNL results in mis-splicing, translocation of CCUG toxic RNA to the cytoplasm, and production of the poly(LPAC) RAN protein and its corresponding pathogenic contributions to DM2. By contrast, the poly(QAGR) RAN protein is not regulated by MBNL1 and it plays a role at least in the brain in DM2. The data supporting linkage of RNA gain-of-function and RAN protein-mediated pathology, and the differential localization of sense and antisense strand products within the brain, provide new insights into understanding the mechanisms underlying the neurologic components of DM2. These findings also support an RNA sequestration failure model as the mechanism for the action of toxic RAN proteins in the brain of patients with DM2.


RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2.
Zu T, Cleary JD, Liu Y, Bañez-Coronel M, Bubenik JL, Ayhan F, Ashizawa T, Xia G, Clark HB, Yachnis AT, Swanson MS, Ranum LPW.
Neuron. 2017 Sep 13;95(6):1292-1305.e5. doi: 10.1016/j.neuron.2017.08.039.

Interim Results Released for AMO Pharma Clinical Trial

Published on Tue, 09/26/2017

AMO Pharma has been conducting a Phase 2a clinical trial of Tideglusib (also known as AMO-02) for adolescents and adults with congenital myotonic dystrophy. Tideglusib is an antagonist for GSK3β, a cell signaling molecule thought to play a role in the pathogenesis of myotonic dystrophy. The AMO clinical trial is a single-blind study of 400 mg and 1000 mg doses of Tideglusib; single-blind means that those conducting the trial know the dose that each patient received and there was no placebo control included in the study. The study is being conducted at Newcastle University.

AMO recently reported interim data from the first cohort of 8 patients who received the 1000 mg dose of Tideglusib. Small, Phase 2a studies such as this are informative as to whether the candidate therapeutic is safe and well-tolerated. AMO reported that no trial subjects withdrew from the study as a result of adverse events or other issues.

Phase 2a studies also are used as a pilot to determine proof of the scientific concept as well as how various study endpoints perform in a particular patient group. Since there have been no prior clinical trials using novel drugs targeted to the brain in congenital myotonic dystrophy (CDM), multiple endpoint measures were explored to help in selection of endpoints for subsequent, definitive trials. AMO reported that data analysis was still ongoing, but that multiple endpoint measures indicated improvements that were statistically significant. With AMO’s focus on developing a drug to address brain manifestations of CDM, some of the endpoints showing improvement included central nervous system symptoms, autistic features and activities of daily living.

AMO Pharma is scheduled to present findings at the upcoming American Neurological Association meeting in San Diego in October. More complete information may be available after that presentation. Based upon these early stage results, AMO intends to launch a larger, multi-site study of Tideglusib in adolescents and children with CMD.

MDF is pleased to see candidate therapeutics moving forward for myotonic dystrophy and appreciates AMO Pharma’s efforts. To date, their results suggest that the drug does not have safety concerns and the positive data from the exploratory endpoints examined here suggest that it could prove effective for CDM. We await the launch and completion of a definitive, multi-site, placebo-controlled controlled trial, the gold standard for the regulatory authorities, for Tideglusib.

Risk of GI Manifestations in DM1 and DM2

Published on Mon, 09/18/2017

GI Symptoms Are an Important Component of DM Disease Burden

The involvement of gastrointestinal functions in all types of myotonic dystrophy is well documented (see MDF’s summary). Common GI symptoms include difficulties in chewing or swallowing, GI reflux, abdominal or chest pain, gallstones, constipation, diarrhea, impaired/painful bowel movements, and incontinence. GI manifestations have been treated by repurposing existing drugs (e.g., mexiletine, metoclopramide, cholestyramine) or behavioral modifications. Awareness of the types and breadth of GI manifestations from appropriately powered studies is vital to inform patient management.

Report on GI Manifestations from a Registry-based Study

James Hilbert and colleagues have reported findings of the frequency, progression and management of GI manifestations using data from the National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members. The investigators evaluated Gi manifestations in a cohort of adult patients (913 DM1 and 180 DM2) enrolled in a patient-reported registry, analyzing data collected between 2002 and 2016, with annual updates on registrants.

GI Manifestations Represent a Substantial Factor in the Burden of Disease.

GI involvement was already prevalent among registrants at initial data entry, as 79% of DM1 and 77% of DM2 registrants reported one or more manifestations. Less than 2% of DM1 and none of the DM2 patients reported GI involvement as the first sign of their disease. In order of descending frequency, trouble swallowing, acid reflux, and constipation were most commonly reported for DM1, while constipation, acid reflux, and trouble swallowing were most commonly reported for DM2. Abnormal liver problems were in sufficient numbers (6-8% of DM1 and DM2) to potentially be a factor for clinical trials. As in prior reports of gender differences, female sex was associated with a higher frequency of GI manifestations in both DM1 and DM2 (constipation and gallbladder problems in this study)Analysis of management practices for GI manifestations included 59 medications used in DM1 and 28 medications in DM2. 

Analysis of disease progression in this studied was based on the approximately 50% of DM1 and DM2 registrants with data available at baseline and year 5. For DM1, trouble swallowing (the most frequent symptom at baseline) and acid reflux were the most frequently reported manifestation not previously present at baseline. Likewise, for DM2, manifestations of constipation and swallowing (the first and third most frequent symptoms, respectively, at baseline) were most frequent new reports at year 5.

Better Understanding of Causes, Consequences and Treatment of GI Manifestations Will Require International Collaboration

The research team notes that GI manifestations have complicating consequences for disease management and patient quality of life. Disease duration was associated with GI manifestations, confirming prior findings in DM1. The pathogenic mechanisms and putative biomarkers for many of the GI manifestations are poorly understood—this group noted no association with repeat length—and treatments are symptomatic. Often symptomatic treatments haven’t been systematically studied in DM. Finally, these researchers note the importance of data sharing to assess GI manifestations, and their progression and treatment, across a larger, better powered cohort.


High frequency of gastrointestinal manifestations in myotonic dystrophy type 1 and type 2.
Hilbert JE, Barohn RJ, Clemens PR, Luebbe EA, Martens WB, McDermott MP, Parkhill AL, Tawil R, Thornton CA, Moxley RT 3rd; National Registry Scientific Advisory Committee/Investigators.
Neurology. 2017 Aug 30. pii: 10.1212/WNL.0000000000004420. doi: 10.1212/WNL.0000000000004420. [Epub ahead of print]


Current Status of Brain Imaging in DM1

Published on Mon, 09/18/2017

Understanding of the CNS Manifestations Is an Unmet Need in DM

While skeletal muscle biomarkers and clinical endpoints are rightly the current focus of interventional clinical trials in DM1, as they are most likely to inform go/no-go decisions, the CNS burden in DM is considerable and likely requires targeted therapy development programs. Pharmaceutical and biotechnology firms increasingly recognize the need for CNS biomarkers and clinically meaningful endpoint measures, but, likely due to the costs of brain imaging studies, efforts to evaluate brain structural changes with MRI are often modest. A recent review details accomplishments in brain imaging in DM and assesses the path forward to more informative studies.

CNS Imaging Studies May Yield Vital DM1 Clinical Trial Endpoint Measures

Dr. Kees Okkersen and team members at Radboud University Medical Centre and the University of Glasgow have conducted a comprehensive review of published studies that used a variety of imaging methodologies (MRI, functional MRI, MRS, ultrasound, SPECT, PET, and CT) to assess DM1. Their review article in Neurology relies upon a total of 81 cross-sectional and longitudinal studies to draw conclusions about the pattern of changes in the CNS in DM1, to show how they relate to other genetic and clinical parameters, and to provide direction to optimize future studies.

The research team followed a careful search/selection strategy that triaged publications from 1974-2016 in the Embase and MEDLINE databases to include 81 studies, reporting a total of 1,663 DM1 cases, in their analysis. Conclusions were drawn from aggregate analysis of findings from these studies and included comments on the strengths, weaknesses, and overall validity of the various imaging strategies used in DM1. The aggregate data showed widespread structural changes to gray and white matter in cerebral cortex, cerebellum, and basal ganglia, with little evidence of specific regional involvement or sparing; a finding consistent with neuropathologic observations in DM1. Substantial white matter involvement was a consistent feature across studies (with prevalence of 70% in DM1 subjects vs. 6% in controls). Specificity in cortical region involvement was seen across the 7 PET studies that met inclusion criteria; these findings were supported by SPECT studies (n = 5). Findings of fMRI studies supported personality and social cognition patterns seen in DM1.

Overall, the aggregate analysis supported some correlations between findings from brain imaging and genetic/clinical features. If clinical trial endpoints are to be developed, it is essential to understand the natural history of the structural and functional changes in the brain in DM1. The research team, however, observed that only 3 of the 81 studies that qualified for their analysis were longitudinal imaging studies. Such studies were deemed to be particularly important since some of the imaging changes in DM1 are associated with normal aging and it will be important to understand whether DM1 pathophysiology starts within specific brain regions before generalizing. 

Taken together, this review of brain imaging provides careful selection and analysis of completed studies in DM1. Sufficiently powered longitudinal studies represent a clear need for the field. Given the high costs of such studies, a consortium approach with an agreed upon data collection, sharing, and analysis protocol is likely the best path forward to understanding and developing therapies for CNS manifestations of DM1.


Brain imaging in myotonic dystrophy type 1: A systematic review.
Okkersen K, Monckton DG, Le N, Tuladhar AM, Raaphorst J, van Engelen BGM.
Neurology. 2017 Aug 2. pii: 10.1212/WNL.0000000000004300. doi: 10.1212/WNL.0000000000004300. [Epub ahead of print] Review.


Understanding DM1 Patient Perceptions about Their Disease

Published on Mon, 09/18/2017

Impact of DM1 on the Brain

DM1 is characterized by involvement of multiple organ systems, raising challenges for understanding disease mechanisms, development of effective disease management strategies, and designing effective and testing therapeutics. The disease burden and unmet need represented by the CNS sequella of DM1 have received insufficient attention to date. Recent efforts by MDF, including the Consensus-based Care Recommendations for Adults with DM1 and the recent session at the 2017 MDF Annual Conference, “Bringing the Patient Voice to CNS-Targeting Drug Development in Myotonic Dystrophy” are important efforts to address this challenge. Given the impact on the brain, a key part of the challenge is understanding how aware are patients of their own disease and to what extent can they assist researchers in improving management and therapy of DM1?

A New Study of Disease Awareness in DM1

Dr. Sigrid Baldanzi and colleagues at the University of Pisa have published results from a cross-sectional study of 65 adult-onset DM1 patients, assessing cognitive function and quality of life. Specifically, the research team was interested in determining how cognitive dysfunction and neuropsychological manifestations of DM1 impacted the patient’s awareness of their own disease—a phenomenon known as anosgnosia or lack of insight.

Anosgnosia, and the consequent reduced patient participation with caregivers, can impact DM1 in multiple ways-- diagnosis can be delayed, lack of awareness and misattribution of the causes of their symptoms can occur, and patient compliance with treatment impacted. To better understand patient awareness in DM1, the research team used a battery of endpoints to assess clinical and neuropsychological status, quality of life, and disease unawareness. The team defined disease unawareness as “an altered ability to recognize the presence or appreciate the severity of deficits in sensory, perceptual, motor, affective, or cognitive functioning.”

The cognitive profile of study subjects was heterogeneous, but executive functions, cognitive flexibility, conceptualization, and visuo-spatial memory tasks ranked below matched control subjects. Assessment of quality of life using INQoL revealed only a mild impact of the patient’s disability. In assessing patient awareness of the impact of their disease, those with mild motor involvement frequently understated their degree of motor impairment in comparison to physician-rated (MIRS) motor abilities. For approximately half of the cohort, discrepancies were noted between patient INQoL ratings and caregivers reports of disease impact. Patients underreported psycho-social difficulties, including their independence and social relationships, when compared to caregiver reports. Patients did not exhibit deficits in awareness of emotional aspects as detected by INQoL.

The research team concluded that DM1 patients did not experience generalized disease unawareness, but rather that unawareness of particular psychosocial and behavioral deficits were present in over half of their cohort. They hypothesize a linkage between anosognosia and brain dysfunction in DM1 and suggest that the terminology “social cognition dysfunction” captures the disease unawareness seen here. Indeed, understanding of the specific features of anosognosia in DM1 may help elucidate its underlying neuroanatomical correlates.  

Finally, the research team noted that analytic tools for anosognisia must be tailored for specific diseases and suggest that patient/caregiver discrepancies in INQoL scores form the basis for such a tool for DM1.


Disease awareness in myotonic dystrophy type 1: an observational cross-sectional study.
Baldanzi S, Bevilacqua F, Lorio R, Volpi L, Simoncini C, Petrucci A, Cosottini M, Massimetti G, Tognoni G, Ricci G,Angelini C, Siciliano G.
Orphanet J Rare Dis. 2016 Apr 4;11:34. doi: 10.1186/s13023-016-0417-z.


Made to Measure: Developing Clinical Tools to Capture the Severity and Progress of DM

Published on Tue, 08/15/2017

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.

Modifying Gene Editing Technology for DM

Published on Tue, 08/15/2017

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.