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 (https://ncats.nih.gov/files/agm-factsheet.pdf).

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.

References:

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

Enhanced risk of DM1 patients developing benign and malignant cancers has been identified in recent registry-based studies (UK Myotonic Dystrophy Patient Registry and National Registry of Myotonic Dystrophy and Facioscapulohumeral Muscular Dystrophy Patients and Family Members). Although sample size was small, one of these earlier studies (Gadalla et al., 2017a) showed elevated risk of skin tumors with the key risk factor, sun exposure, similar to that of the general population.

New Longitudinal Data on Skin Cancer Risk in DM1

Dr. Shahinaz Gadalla and colleagues at the U.S. National Cancer Institute, UK Medicines and Healthcare Products Regulatory Agency, Queen’s University Belfast, and Newcastle University have published a large electronic medical records study of cancer risks in DM1 that included 1,061 DM1 patients and 15,119 DM-free subjects (Gadazlla et al. 2017b). After controlling for a variety of variables, the research team identified a cohort that included 35 DM1 and 108 DM-free individuals who developed skin cancer; incidence rates were approximately 435/100,000 for DM1 and 131/100,000 for DM-free subjects. DM1 patients had significantly increased risk for developing skin cancer (all types combined), with the highest risk for basal cell carcinoma. A 2-fold increase in risk of melanoma among DM1 patients did not reach significance.

Overall, a DM1 diagnosis was associated with approximately 6-fold excess risk of developing non-melanoma skin cancers and as much as 7-fold excess risk for all skin cancers combined. There were no gender or age at diagnosis differences in skin cancer risk among DM1 patients.

Clinical Care Guidance and Future Studies

Taken together, data from recent registry-based studies suggest an elevated risk of skin cancer in DM1 patients, with an addressable key risk factor—patients should minimize ultraviolet light exposure and seek medical opinion for suspicious skin lesions. This latest study assessed a large cohort, used a longitudinal design and was carefully controlled, lending a high degree of confidence to these findings. Biologic mechanisms underlying the increased cancer risk in DM1 are currently unknown—potential relationships to MBNL depletion or mis-splicing of skin-related genes should be probed.

References:

Pigmentation phenotype, photosensitivity and skin neoplasms in patients with myotonic dystrophy.
Gadalla SM, Hilbert JE, Martens WB, Givens S, Moxley RT 3rd, Greene MH.
Eur J Neurol. 2017a May;24(5):713-718. doi: 10.1111/ene.13276. Epub 2017 Mar 20.

Risk of skin cancer among patients with myotonic dystrophy type 1 based on Primary care physician data from the United Kingdom Clinical Practice Research Datalink.
Wang Y, Pfeiffer RM, Alsaggaf R, Meeraus W, Gage JC, Anderson LA, Bremer RC, Nikolenko N, Lochmuller H, Greene MH, Gadalla SM.
Int J Cancer. 2017b Nov 7. doi: 10.1002/ijc.31143. [Epub ahead of print]

Sudden cardiac death resulting from atrioventricular block or ventricular arrhythmias is major risk factor (2^nd leading cause of death after respiratory failure) for patients with DM1. These recommendations include annual ECG and use of electrophysiology testing when there is the potential for serious conduction block and arrhythmias. Developing sensitive biomarkers with the potential for early identification of those at risk for cardiac events is a critically important need for care of patients living with DM1.

Cohort Study Assessing Biomarkers for Cardiac Abnormalities

Prof. Raffaele Bugiardini and colleagues at the IRCCS Policlinico San Donato and University of Bologna evaluated serum biomarkers (high-sensitivity cardiac troponin T (hs-cTnT), high-sensitivity cardiac troponin I (hs-TnI), creatine kinase (CK) and N-terminal pro B-type natriuretic peptide (NT-proBNP) in 60 study subjects, 46 with genetic confirmation of DM1 and 14 with DM2. These serum measures were correlated with ECG and left ventricular ejection fraction (LVEF) determined by echocardiography.

Screening for Prospective Serum Biomarkers

ECG abnormalities were detected in 39% of patients. All but 2 patients had normal LVEF. Elevations of hs-cTnT and CK were observed in 92% of DM subjects, while hs-cTnI levels were within normal limits. Higher levels of hs-cTnT were found in patients with ECG measures above the normative range., but, after adjusting for age, gender, NT-proBNP levels and CK, hs-cTnT levels did not correlate with ECG parameters. Twelve of the 60 subjects (9 DM1 and 3 DM2) had elevated NT-proBNP levels.  Measurements of NTpro-BNP > 125 pg/ml were an independent predictor of ECG abnormalities.

Outcomes

The research team noted that NT-proBNP is released during cardiac repair and that it is not yet known how elevated levels may relate to the development of conduction system abnormalities in DM. They do show that NT-proBNP levels > 125 pg/ml correlates with the presence of conduction abnormalities in DM, regardless whether the molecular diagnosis was DM1 or DM2. Identification and validation of an NT-proBNP level that would trigger stricter clinical monitoring and determination of the relationship between NT-proBNP levels and the severity of ECG abnormalities awaits study of a larger cohort.

Reference:

Cardiac involvement in myotonic dystrophy: The role of troponins and N-terminal pro B-type natriuretic peptide.
Valaperta R, De Siena C, Cardani R, Lombardia F, Cenko E, Rampoldi B, Fossati B, Brigonzi E, Rigolini R, Gaia P, Meola G, Costa E, Bugiardini R.
Atherosclerosis. 2017 Oct 21;267:110-115. doi: 10.1016/j.atherosclerosis.2017.10.020. [Epub ahead of print]

Cutting Through the Hype

As if we needed actual data to understand that CRISPR technology is a hot topic—a PubMed search on “CRISPR” yields over 7,000 hits, while the NIH RePORTER database search returns 2,316 active research grants with reference to the topic. Potentially transformative therapeutic strategies have always been hyped, but the explosion in the mass media coverage makes it difficult to believe that anyone remains who has not heard the term CRISPR and the unfortunate implication that marketed therapies are “imminent.”

Our task in the scientific, pharma/biotech and advocacy communities is to counter the therapeutic misconception that CRISPR will be a panacea for any inherited disease, while taking pragmatic steps to address the not inconsequential barriers of rigorous efficacy studies, scale-up production, delivery/exposure, efficiency, and even ethics to optimize and evaluate the potential of this important new tool.

Finding the Optimal CRISPR Targets

Initial efforts to apply CRISPR/Cas9 approaches to DM focused on reducing/removing expanded CTG tracts from the DMPK gene. An additional challenge for gene editing in expanded repeat disorders is the need to remove large DNA tracts. Indeed, Dr. Bé Wieringa and colleagues found that removal of expanded CTG repeats was feasible, but required cutting from both sides of the repeat to avoid increasing the genomic instability that drives DM (see http://www.myotonic.org/gene-editing-dm). DNA targeting may ultimately prove to be the optimal approach for DM, but more developmental and mechanistic work is required to facilitate in situ removal of large expansions safely and efficiently.

Within the CRISPR field, the approach of targeting RNA has attracted considerable interest. Two recent papers from Dr. Feng Cheng’s lab (Broad Institute of MIT and Harvard) have demonstrated RNA targeting using CRISPR/Cas13.  In the first (Cox et al., 2017), they evaluated programmable editing of RNA transcripts to alter coding and thereby correct mutations at the level of the transcript (a system they designate as RNA Editing for Programmable A to I Replacement or REPAIR). This system is designed to address single base mutations and thus may have limited to no applicability in repeat expansion disorders, although continued attention to the associated technological advances is warranted. The second publication (Abudayyeh et al., 2017) demonstrates that CRISPR/Cas13a has the potential for targeted knockdown of RNA, with similar efficiency to and better specificity than RNAi. Such an approach is comparable to knockdown of DMPK transcripts with antisense oligonucleotides that recruit RNaseH mechanisms.

To Target RNA or DNA in DM?

Previously, Dr. Gene Yeo’s group showed that RNA-targeted Cas9 in an in vitro model degrades toxic DMPK transcripts, increases in the MBNL protein, and reduces or eliminates the gene splicing defects that characterize DM (see http://www.myotonic.org/modifying-gene-editing-technology-dm).

In a recent paper in Molecular Cell (Pinto et al., 2017), Dr. Eric Wang and colleagues (University of Florida) reported that deactivated Cas9 (dCas9) specifically binds expanded repeat tracts at the DMPK locus and impedes their transcription. They evaluated multiple CTG repeat lengths and showed that dCas9 coupled to a guide RNA achieved knockdown in a repeat length-dependent manner, with binding efficiency and potency of knockdown increasing with the number of repeats. Moreover, the group showed that the dCas9-gRNA strategy reduces nuclear foci, restores normative splicing, and blocks RAN translation in both HeLa cell models and DM1 patient myoblasts and in skeletal muscles of systemically injected HSA-LR mice.

Next Steps for DM

Efforts to target the expanded repeat DMPK RNA or DNA have shown potential as a therapeutic strategy, but it must be recognized that these efforts are at a preclinical proof of concept stage. Both approaches face questions of delivery, specificity of targeting and reagent efficiency once on target that are likely to become more complex when CRISPR as a candidate therapeutic transitions to clinical testing. For example, if AAV is used for CRISPR reagent delivery, the tissue targeting specificity inherent to many AAV serotypes must be overcome if the multi-systemic consequences of DM are to be addressed.

Hype is a poor substitute for pragmatic scientific progress. As we go forward, all parties are cautioned to avoid language that feeds therapeutic misconception—mouse studies are purely experiments in a so-so disease model—the mice are neither treated nor cured; clinical trials also are experiments, not treatments, until the agent receives regulatory approval. Finally, recruitment of substantive funding and scientific expertise to further optimize and test the RNA and DNA targeting strategies for DM is essential.

References:

RNA editing with CRISPR-Cas13.
Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F.
Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180. [Epub ahead of print]

RNA targeting with CRISPR-Cas13.
Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang F.
Nature. 2017 Oct 12;550(7675):280-284. doi: 10.1038/nature24049.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.
Pinto BS, Saxena T, Oliveira R, Méndez-Gómez HR, Cleary JD, Denes LT, McConnell O, Arboleda J, Xia G, Swanson MS, Wang ET.
Mol Cell. 2017 Oct 18. pii: S1097-2765(17)30711-6. doi: 10.1016/j.molcel.2017.09.033. [Epub ahead of print]

Genetics and Epigenetics of CDM

Congenital myotonic dystrophy (CDM) is typically maternally inherited and associated with CTG expansions of greater than 1000 repeats. A previous report by Drs. Karen Sermon and Christopher E. Pearson (Barbé et al., 2017) suggested that repeat length alone was not sufficient to trigger CDM, but that co-occurrence of specific epigenetic changes upstream of the DMPK locus, CpG hypermethylation, was an essential factor in both the phenotypic severity and maternal inheritance pattern characteristic of CDM (see http://www.myotonic.org/inheritance-cdm). Conceptually, it may not be appropriate to think of CDM as ‘severe DM1,’ but rather as a mechanistically distinct disease also localized to the DMPK locus. Achieving clarity on genetic and epigenetic mechanisms in CDM should inform testable hypotheses as to downstream events responsible for the CDM phenotype and putative targets for therapy development.

Pathogenic Mechanisms Underlying CDM

Dr. Masayuki Nakamori (Osaka University Graduate School of Medicine) and colleagues recently published studies of downstream signaling pathways in CDM, focusing on molecular events that may contribute to the skeletal muscle immaturity seen in CDM but not DM1 or DM2. Through study of CDM patient skeletal muscle samples, the group established a correlation between expanded CUG tracts, methylation of CpG islands upstream of the DMPK locus, and skeletal muscle immaturity. The aberrant methylation, in turn, correlated with dysregulation of transcription in both sense and antisense directions and concomitant increase in expanded CUG repeat transcript toxicity.  The team linked RNA toxicity to PKR activation and increased ER stress response, NF-κB activation and chronic activation of IL-6/STAT3 myokine signaling that promotes myocyte proliferation/delays differentiation.  The authors propose an integrated disease model and suggest that over-activation of IL-6 myokine counters it’s positive role in muscle development and instead is responsible for the profound failure of skeletal muscle maturation and, over time, muscle atrophy in CDM.

Some components of the signaling pathway described by the research team appear to be restricted to CDM.  While activation of ER stress response was also observed in DM1, the IL-6 myokine signaling activation found in CDM muscle samples is not detected in skeletal muscles of either DM1 patients or HSA-LR mice. The authors note that a mechanistic linkage between very large expanded CUG repeats in DMPK and CpG methylation status is not yet resolved. Given the potential importance of hypermethylation at sites upstream of DMPK in differentiating CDM from DM1, and in the maternal inheritance pattern of CDM, understanding how repeats > 1000 impact CpG methylation remains an important question.

Implications for CDM

Elucidation of pathogenic mechanisms in CDM, particularly downstream signaling pathways essential to disease onset and progression, increases understanding of the disease as well as provides novel targets for therapy development. Maturational failure of skeletal muscle represents a key contributor to the burden of disease in CDM. The downstream pathways described by this research team appear to include tractable targets for therapeutic intervention, such as NF-κB, STAT and IL-6, which represent drug targets under investigation for other disease classes, including oncology and inflammatory disorders, raising prospects of repurposing drugs already under development for DM.

Reference:

Aberrant Myokine Signaling in Congenital Myotonic Dystrophy.
Nakamori M, Hamanaka K, Thomas JD, Wang ET, Hayashi YK, Takahashi MP, Swanson MS, Nishino I, Mochizuki H.
Cell Rep. 2017 Oct 31;21(5):1240-1252. doi: 10.1016/j.celrep.2017.10.018.

Understanding Heterogeneity in DM

Evaluation of large cohorts in the DM1-Scope registry, established the impact of the variables of age and gender in DM1 (De Antonio et al., 2016; Dougan et al., 2016). Men are more likely to experience ‘traditional’ muscular system-related DM1 symptoms (myotonia, muscle weakness, cardiac and respiratory involvement), while other symptoms are more likely in women (cataracts, dysphagia, digestive tract dysfunction, incontinence, thyroid disorder and obesity). Likewise, age of onset has clear implications for DM1 symptom symptom occurrence and chronology. By contrast, the impact of the variables of age and gender is less well understood for DM2, but remains a critically important question due to the heterogeneity of the disease.

A Retrospective Study Informs the Natural History of DM2

Prof. Benedikt Schoser (Ludwig-Maximilians-University of Munich) and colleagues have explored variables in disease symptomatology in a retrospective study of a 307-patient cohort with genetically confirmed DM2 from the German Registry for Myotonic Dystrophy.

Study subjects showed mean onset at 42 years of age; most had a single symptom at onset, with proximal weakness, myalgia and myotonia the most frequent symptoms (in order of descending frequency). Weakness at disease onset was associated with older age. New neurological examinations (at an mean age of 56 years), showed 77% of the cohort with mild to moderate weakness of at least one muscle group. In decreasing order of frequency, multisystem involvement included cataract, hyperlipidemia, hypertension, thyroid disorders, affective disorders, and cardiac disease (implanted ICDs/pacemakers were frequent). A previously reported pattern of altered laboratory findings was confirmed in this DM2 cohort: hyper-CKaemia, dyslipidaemia, elevated GGT, low IgG, and elevated ALT and AST.

Age at onset of DM2 and gender were significantly and independently associated with specific patterns of symptoms. Advancing age was associated with increasing number of organ systems involved, but decreasing odds that a subject would develop either myotonia or myalgia. Women had a higher total number of organ systems involved.

A high percentage (87%) of study subjects had myotonia (evaluated by emg), although no gender differences were detected. In contrast to findings in DM1, muscle weakness was more common and more severe in women at time of diagnosis, while pain/myalgia was most common in men. As in DM1, cataract and thyroid and gallbladder diseases were observed more frequently in women. Women with DM2 also more frequently needed waling aids.

Patients experiencing early onset of DM2 were more likely to have broader organ system involvement.

Age and Gender Impact the DM2 Phenotype

Taken together, the research team observed that age and gender have a profound impact on the DM2 phenotype. Both female gender and age appear to contribute toward a greater burden of disease in DM2. Analysis of ancestry of patients in the registry suggested that a DM2 founder mutation may have arisen in the Upper/Lower Silesia region of Poland, Germany and the Czech Republic.
 

Reference:

Assessing the influence of age and gender on the phenotype of myotonic dystrophy type 2.
Montagnese F, Mondello S, Wenninger S, Kress W, Schoser B.
J Neurol. 2017 Oct 30. doi: 10.1007/s00415-017-8653-2. [Epub ahead of print]

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.

Reference:

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.

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.

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.