Originally presented on November 15th, 2024.

Do you have questions for myotonic dystrophy (DM) doctors and therapists? Join Dr. Lorelei Thornburg, Obstetrician/Gynecologist, and Dr. Johanna Hamel, Assistant Professor in Neurology, Pathology, and Laboratory Medicine, from the University of Rochester for an "Ask the Expert" webinar on reproductive health and DM!

Find all our upcoming Ask the Expert dates and previously recorded sessions! >>>

MDF Resources referenced in this video:

• Clinical Recommendations for People of Pregnancy Potential with Myotonic Dystrophy
• Toolkits & Publications
• Support Groups
• Calendar of Events
• DM Drug Development Pipeline
• Myotonic Dystrophy Family Registry
• 2025 MDF Annual Conference
• Sign up for My Dispatch, our monthly e-newsletter!

About our Myotonic Dystrophy Experts

Dr. Lorelei Thornburg, Obstetrician/Gynecologist, University of Rochester  

Dr. Thornburg joined the faculty of the University of Rochester in June 2008. She received her undergraduate degree from Kalamazoo College graduating cum laude, and her medical degree from Wayne State University in Detroit, Michigan where she received the David S. Diamond Memorial Award in Obstetrics and Gynecology and was elected to the Alpha Omega Alpha honor society. She completed her residency in Obstetrics and Gynecology at the University of Rochester in Rochester, New York in 2005, where she also served as administrative chief resident during the final year of her residency. Dr. Thornburg completed her fellowship in Maternal Fetal Medicine (Perinatology- High Risk Obstetrics) at the University of Rochester in Rochester, New York in 2008. She received the Ward L Ekas, the George C. Trombetta, the Obstetrical Perinatology, Dr. Curtis J. Lund, and the Creog Awards for leadership and teaching during the course of her residency, and the Todd Faculty Fellowship in Maternal Fetal Medicine Award during her fellowship. She is a board-certified Obstetrician/Gynecologist and Maternal Fetal Medicine and is a Fellow of the American College of Obstetrics and Gynecology. Active areas of research include maternal obesity, ultrasound and resident education.

Dr. Johanna Hamel, Assistant Professor in Neurology, Pathology, and Laboratory Medicine, University of Rochester  

Dr. Hamel is an Associate Professor of Neurology, Pathology and Laboratory Medicine and specializes in the diagnosis and treatment of neuromuscular diseases. Dr. Hamel cares for patients with acquired and hereditary neuromuscular diseases in clinic, with special expertise in myotonic dystrophy (DM) type 1 and type 2 and FSHD. Dr. Hamel also performs electrodiagnostic studies in the EMG lab and teaches residents about neuromuscular diseases. She graduated from medical school at the Martin-Luther-University Halle-Wittenberg in Germany and worked as a neurology resident and researcher at the Charité in Berlin before completing a neurology residency and a clinical neuromuscular fellowship at the University of Rochester.

Join Certified Genetic Counselor Tiffany Grider from the University of Iowa for an recorded webinar on genetic testing. Topics include:

+ The underlying genetic cause of myotonic dystrophy type 1.
+ How this genetic cause leads to so many different medical problems including muscle weakness.
+ Inheritance, the risks for congenital myotonic dystrophy and the genetic basis for the more severe symptoms.
+ Different types of genetic testing including diagnostic, presymptomatic, and prenatal.
+ Research being done for gene therapies.

View Tiffany Grider's clinical profile.

Download the slides (.pptx).

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]

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.