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.

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.

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

Cardiac troponin-I, a sarcomeric regulatory protein integral to skeletal and cardiac muscle contraction, has long been utilized as a diagnostic and prognostic biomarker of heart disease. For muscular dystrophies, elevated serum creatine kinase and troponin are associated with myopathic changes in muscle. Understanding the sensitivity of the analytical tools, as well as the types of cardiac issues that may result in elevated cardiac markers in serum, is critical to use of these assays in monitoring myotonic dystrophy type 1 (DM1) patients.

The constellation of cardiac involvement in DM1 includes atrioventricular block, prolonged QT interval, prolonged QRS interval, increased ventricular premature contractions, atrial fibrillation/flutter, right/left bundle branch block, non-sustained ventricular tachycardia and left ventricular systolic dysfunction (Petri et al., Int. J. Cardiol. 160: 82-88, 2012). Prior reports identified a correlation between CTG repeat length and cardiac dysfunction and linked the degree of neuromuscular and cardiac involvement in patients.

A large multi-center study in Scotland recently reported out an analysis of serum levels of cardiac troponin-I (cTnI) in a cohort of 117 well-characterized DM1 patients recruited from outpatient clinics. Nine subjects had cTnI levels that exceeded the 99th percentile of the general population. One-third of subjects with elevated cTnI also had left ventricular systolic dysfunction. The authors noted that elevations in cTnI did not correlate with CTG length, were not predictive of severe conduction abnormalities and did not correlate with muscle strength (by MIRS score). There also was no association between cTnI level and the presence of an implanted cardiac device.

Overall, the authors suggest that cTnI levels represent a potential biomarker to assess risks in the management of DM1 patients and for stratification of subjects in clinical trials. Although the lack of correlation of cTnI levels and MIRS score suggests a cardiac origin for elevated serum cTnI, the underlying responsible pathology in the context of known cardiac phenotype of DM1 is currently unclear. Finally, the authors suggest that the overall sample of patients with elevated cTnI is small and propose these findings as exploratory, requiring follow-up of this and other putative cardiac biomarkers in larger cohorts.

Reference:

Elevated Plasma Levels of Cardiac Troponin-I Predict Left Ventricular Systolic Dysfunction in Patients with Myotonic Dystrophy Type 1: A Multicentre Cohort Follow-up Study.
Hamilton MJ, Robb Y, Cumming S, Gregory H, Duncan A, Rahman M, McKeown A, McWilliam C, Dean J, Wilcox A, Farrugia ME, Cooper A, McGhie J, Adam B, Petty R; Scottish Myotonic Dystrophy Consortium., Longman C, Findlay I, Japp A, Monckton DG, Denvir MA.
PLoS One. 2017 Mar 21;12(3):e0174166. doi: 10.1371/journal.pone.0174166.

Myotonic Dystrophy Anesthesia Guidelines

Please know that the use of anesthesia raises special risks to those living with myotonic dystrophy (DM), as the disease results in heightened sensitivity to sedatives and analgesics. Pay particular attention to the serious complications that can arise in the post-anesthesia period, when risk of aspiration and other complications increase. 

MDF has published two versions of its Anesthesia Guidelines:

• A one-page summary of the anesthesia guidelines to share with your clinician and anesthesiologist.
• The complete "Practical Suggestions for the Anesthetic Management of a Myotonic Dystrophy Patient".

Download an electronic copy of the latest versions of both documents on the Toolkits & Publications page.

New to DM? Click here for more information.

February is American Heart Month, and MDF has partnered with Drs. Katharine Hagerman and Marianne Goodwin to highlight important research on cardiac issues in myotonic dystrophy for our community.

Researchers in the lab of William Groh, MD, at Indiana University have been studying heart involvement in myotonic dystrophy type 1 (DM1), including heart conduction defects, cardiac rhythm disturbances, and structural heart abnormalities for some time. The group performed cardiac imaging and analysis of the electrical impulses of the heart by electrocardiography (ECG or EKG) on individuals with DM1, and found that certain structural changes in the heart are increased in individuals with cardiac electrical abnormalities. In addition, the study identified predictors of heart failure and dysfunction, including increased age and changes in electrical conduction of the heart observed by ECG, specifically measurements known as PR interval and QRS duration. These indicators may be used by doctors to help predict which DM patients would benefit most from implantable cardiac rhythm devices such as pacemakers. Additionally, the group found that some individuals with DM1 benefit more from devices known as implantable cardioverter-defibrillators (ICDs) than standard pacemakers to help correct heart rhythm disturbances.

The heart involvement in myotonic dystrophy type 2 (DM2) is similar to that seen in DM1, however controversy exists over the frequency and severity of heart irregularities in the DM2 population. To address this, a research team led by Giovanni Meola, MD, in the Neuromuscular Clinic at IRCCS Policlinico San Donato in Italy compared cardiac abnormalities in DM1 and DM2. Using tests such as ECG and echocardiography, the study found that heart involvement is less common and generally milder in DM2 than DM1. However, individuals with DM1 and DM2 are both at increased risk of heart abnormalities such as cardiac arrhythmias and conduction disturbances. These heart irregularities can be progressive with age, and may be present in individuals even when no symptoms are experienced. Therefore, people living with DM should receive annual cardiac evaluations, including ECG, by primary care physicians or cardiologists to promote heart health.

For more information on heart health in DM, please see our Body Systems Tool.

To learn more about heart symptoms, exams and management, watch MDF's webinar "Cardiac Issues Related to DM," by Dr. William Groh, a leader in the field.

Papers on DM1 heart issues:

• "Pacemaker and Implantable Cardioverter-Defibrillator Use in a US Myotonic Dystrophy Type 1 Population"
• "Prevalence of Structural Cardiac Abnormalities in Patients with Myotonic Dystrophy Type 1"
• "Increased Mortality with Left Ventricular Systolic Dysfunction and Heart Failure in Adults with Myotonic Dystrophy Type 1"

Papers on DM2 heart issues:

• "The Frequency and Severity of Cardiac Involvement in DM2: Long-term Outcomes"

02/26/2015

Tackling DM from Basic Research through Clinical Care

Tetsuo Ashizawa, MD, better known as "Tee" to colleagues and patients, has focused his career on the search for treatments for myotonic dystrophy (DM). As one of seven primary investigators who will participate in the first clinical trial of a potential treatment for DM1, Dr. Ashizawa may be closer than ever to achieving that goal. Yet in addition to pursuing research with dedication and tenacity, he has also been committed to providing the best possible care to people living with DM. Dr. Ashizawa's engagement in myotonic dystrophy spans basic research, translational science, patient-oriented research and clinical care.

Originally trained in neuromuscular diseases, Dr. Ashizawa first became involved in DM as a basic researcher, working with a team at Baylor College of Medicine to hunt for the DM gene. "There were actually several teams working internationally to find the gene," said Dr. Ashizawa. "Interestingly, in 1992 the various research teams all had the same finding, which was identification of DMPK, the genetic mutation responsible for myotonic dystrophy type 1. It was an exciting time, and that was the beginning of our journey to find treatments and a cure."

Patients Play a Key Role with Researchers

In 1998, as Dr. Ashizawa was expanding his research efforts, he received an email that would broaden his perspective. Shannon Lord, the mother of two boys with juvenile-onset DM1, wanted to make a donation to advance DM research. She provided a grant to Dr. Ashizawa through the Hunter Fund, an account named after her older son and established by Shannon and her husband Larry to support DM research projects. The grant was the start of a long-term friendship between Dr. Ashizawa, Shannon and Larry Lord, and ultimately led to a DM scientific meeting organized by Dr. Ashizawa and including the Lord family. "It was so powerful," said Dr. Ashizawa. "Before this meeting, many in the scientific community only saw DM through a microscope. Now investigators could see and understand the human face of the disease. It was a real morale booster for everyone and provided a great deal of momentum to move our work forward."

By then Dr. Ashizawa had also co-founded the International Myotonic Dystrophy Consortium (IDMC) to bring together scientists and clinicians focusing on DM. Shannon Lord attended the third biennial IDMC meeting in Kyoto in 2001, serving in the role of patient advocate and introducing patient advocacy to the IDMC research community. By the fourth meeting, about one hundred patients and families attended, and the participation of a large number of patients at these international meetings has since become routine. Today, IDMC meetings provide a unique opportunity for global researchers, clinicians and patients to come together; IDMC 10 will be held next June in Paris, France. "Without patient involvement, we wouldn't be able to push forward on the research frontier," Dr. Ashizawa said.

Research Moves Out of the Lab

By 2011, DM science had progressed significantly in the development of potential treatments for DM1. Seven research and clinical institutions around the country are currently preparing to launch the first clinical trial in affected patients to test the efficacy of an antisense oligonucleotide (ASO) therapy, DMPKrx, in people affected by DM1. The University of Florida (UF) will serve as one of these sites, with Dr. Ashizawa as the Primary Investigator for the institution.

Dr. Ashizawa has recently started a project looking at DM1 patient-derived, induced pluripotent stem cells (iPSCs), which can be developed into different cell types needed for research, e.g. muscle, heart, or even brain cells. These cells can help researchers understand how DM affects different body systems and causes disease symptoms. While the clinical use of these cells may be a long way off, iPSCs have a more immediate and critical function as a platform for the screening of compounds to find drugs that have therapeutic potential in DM1. "It's a very exciting time in DM research," Dr. Ashizawa says.

Providing Multidisciplinary Care in the Clinic

In addition to his research projects, Dr. Ashizawa oversees the clinical program at the University of Florida. Patients benefit from a multidisciplinary team of doctors that includes cardiologists, anesthesiologists and geneticists. "We help patients access any clinical trials for which they may be eligible," he says. "And when new treatments become available we are committed to helping our patients access them as soon as possible."

Dr. Ashizawa has published over 190 research papers and 35 book chapters. He is currently Executive Director at the McKnight Brain Institute at UF and Professor and Chair of the Department of Neurology at the UF College of Medicine, and he serves on MDF's Scientific Advisory Committee. With Drs. Maurice Swanson and S.H. Subramony, he has recruited Dr. Laura Ranum to UF and is in the process of recruiting a handful of other key DM investigators to build one of the strongest DM research teams in the world. "We are very hopeful about the research and treatment possibilities on the horizon. We have a distance to go and there are many questions to answer, but we won't stop working," says Dr. Ashizawa. "We are dedicated to our patients and to collaborating with them to find a cure."

12/09/2014