Respiratory Care Recommendations for Myotonic Dystrophy Patients During the COVID-19 Pandemic

MDF would like to provide respiratory care recommendations for myotonic dystrophy patients during the COVID-19 pandemic.

We understand that this is a very challenging time and we would like to provide tips for patients, caregivers, and medical providers on the use of noninvasive positive pressure ventilation (NIPPV) if you have been exposed to this virus or have symptoms of COVID-19 infection.

Please consider keeping a copy of these recommendations with you to provide to your medical team if needed.

Download the Respiratory Care Recommendations for Myotonic Dystrophy Patients During the COVID-19 Pandemic.

Les directives spécifiques pour les patients atteints de dystrophie myotonique et leurs médecins en cas d’infection par COVID-19, crées par Myotonic et traduites par NMD4C, peuvent être téléchargées ICI.

We encourage you to advocate strongly for yourself if you begin to feel ill and need to interact with a medical provider. Let them know you have myotonic dystrophy and that these guidelines were developed by leading pulmonologists and neurologists specifically for myotonic dystrophy patients suspected of being infected with COVID-19.

Please remember to follow CDC guidelines to help stay healthy and to first call or use telemedicine if you have questions for your doctor. These steps will help you avoid unnecessary trips to the hospital that may place you at increased risk of exposure to the virus.

Be well and healthy,

The MDF Team

Evidence-based Guidelines

Due to the multisystemic nature of myotonic dystrophy type 1 (DM1), it has been a challenge to conduct the studies and collect the rigorous evidence required to create an evidence-based guideline for the cardiac clinical care of patients with myotonic dystrophy type 1. In order to improve the quality of life and standardize care for people living with DM1, MDF organized and supported 10 leading DM1 cardiologists in Canada, Japan, Western Europe, the United Kingdom, and the United States to create the Consensus-based Care Recommendations for Cardiologists Treating Adults with Myotonic Dystrophy Type 1.

Publication Preparation

To promote the development of cardiac care guidelines for myotonic dystrophy, MDF solicited the input of care experts and organized the drafting of these recommendations. The 10 leading DM1 cardiologists created the Consensus-based Care Recommendations for Cardiologists Treating Adults with Myotonic Dystrophy Type 1 through an iterative drafting process. All authors contributed content to create the first draft and leading author Elizabeth McNally, M.D., Ph.D. was in charge of managing further editing cycles. The process began in 2017 and concluded in February 2020, with the article published on the Journal of the American Heart Association website on 2/6/20 and in print on 2/18/20 in Volume 9, Issue 4.

Tell Your Cardiologist

The guidelines review cardiac management of myotonic dystrophy, including surveillance for arrhythmias and left ventricular dysfunction, both of which occur in progressive manner and contribute to morbidity and mortality. Other topics reviewed in the guidelines include heart imaging, cardiomyopathy treatment and stroke risk, concluding in 15 summary recommendations. The recommendations represent expert consensus opinion from those with experience in the management of myotonic dystrophy, in part supported by literature‐based evidence where available.

The guidelines are intended for use by cardiologists but individuals diagnosed with DM1 are encouraged to provide a copy of the recommendations to their cardiologists. The guidelines represent an exciting step in the cardiac treatment and management of DM1 and MDF is thrilled to support this vital educational effort to standardize care.

Resources

Digitally access the full article on the Journal of the American Heart Association website.

You can download the full Consensus-based Care Recommendations for Cardiologists Treating Adults with Myotonic Dystrophy Type 1 as well as other critical care resources to share with your doctor on our Toolkits & Publications page.

If you would like a hard copy of the article or guidelines mailed to you, please email us at info@myotonic.org.

References:

Clinical Care Recommendations for Cardiologists Treating Adults With Myotonic Dystrophy
McNally EM, Mann DL, Pinto Y, Bhakta D, Tomaselli G, Nazarian S, Groh WJ, Tamura T, Duboc D, Itoh H, Hellerstein L, and Mammen PPA
JAHA. 2020 Feb 6. doi 10.1161/JAJA.119.014006 [Epub ahead of print]

SAN FRANCISCO, CA (May 2, 2019): MDF is pleased to announce the publication of the first-ever Consensus-based Care Recommendations for Congenital and Childhood-onset Myotonic Dystrophy Type 1 and Myotonic Dystrophy Type 2. The Quick Reference Guides to the full Consensus-based Care Recommendations were published in Neurology Clinical Practice online April 24th. The articles will be published in the NCP print journal in Summer 2019. MDF is working internationally to help ensure that the care recommendations are disseminated and adopted broadly.

Currently no evidence-based guidelines exist to establish standards of care for myotonic dystrophy, and comprehensive care guidelines will not be available until a number of studies are conducted to establish the rigorous evidence needed to create them. As a result, patients and caregivers report difficulty accessing informed clinicians and quality care.

To develop the recommendations, eleven international clinicians experienced in the care of infants and children living with congenital and childhood-onset DM1, and fifteen international clinicians experienced in the care and treatment of adults with DM2 worked collaboratively for over a year to develop consensus regarding care strategies for over 20 different body systems. The resulting clinical care recommendations are intended to help standardize and elevate care for people living with myotonic dystrophy, improving quality of life for affected families and reducing variability in clinical trial and study environments.

The publications and clinical care recommendations are accessible via the links below.

Consensus-based Care Recommendations for Adults with Myotonic Dystrophy Type 2

• Publication - Consensus-based Care Recommendations for Adults with Myotonic Dystrophy Type 2
• Comprehensive document and Quick Reference Guide - Consensus-based Care Recommendations for Adults with Myotonic Dystrophy Type 2

Consensus-based Care Recommendations for Congenital and Childhood-onset Myotonic Dystrophy Type 1

• Publication: Consensus-based Care Recommendations for Congenital and Childhood-onset Myotonic Dystrophy Type 1
• Comprehensive document and Quick Reference Guide - Consensus-based Care Recommendations for Congenital and Childhood-onset Myotonic Dystrophy Type 1

Adults with Myotonic Dystrophy Type 1

Clinical care recommendations for adults with myotonic dystrophy type 1 were published in Fall 2018. Access the comprehensive document and Quick Reference Guide here.

About Myotonic Dystrophy

Described as “the most variable of all diseases found in medicine”, myotonic dystrophy (DM) is an inherited disorder that can appear at any age and that manifests differently in each individual. The most common form of adult-onset muscular dystrophy, DM1 population-based prevalence is 1:2,300 people worldwide (Johnson 2018), and can cause muscle weakness, atrophy and myotonia, as well as problems in the heart, brain, GI tract, endocrine, skeletal and respiratory systems. There is currently no treatment or cure for DM.

About MDF: 

MDF is the world’s largest DM patient organization. Its mission is My Cause. My Cure: is to enhance the lives of people living with myotonic dystrophy, and advance research efforts focused on finding treatment and a cure for this disorder through education, advocacy and outreach.

For additional clinical resources offered by MDF, click here.

Nationwide Children’s Hospital and Ohio State University have operated a 5-day myology training course for the last seven years. The course includes common lectures in the mornings and separate clinical and laboratory tracks in the afternoons. MDF staff have participated as course lecturers in the past and attest to the value of the course. Trainees accepted for the course pay only for travel and some meals, as there is no cost for the course itself or for local lodging. Attendance is capped at 60 (30 in each track), so those interested need to register quickly.

A slightly shortened version of the course announcement from Dr. Kevin Flanigan is provided here.

“I would like to invite you to send your trainees to the eighth annual Nationwide Children’s Hospital/Ohio State University Myology Course, to be held at Nationwide Children's Hospital from Monday, August 26th through Friday, August 30, 2019. The goal of this course is to provide trainees with an up-to-date and expert survey of neuromuscular diseases. The expert faculty will address clinical syndromes, mechanisms of pathogenesis, molecular therapeutics, and aspects of career development.

“The course is available to both pre-and post-clinical trainees, and both clinical- and laboratory-based trainees are welcome. We have structured the course so that both groups will be together for morning lectures.

“The Clinical Track is primarily directed at clinical fellows (in Neuromuscular Disease; Genetics; Electrophysiology; Physical Medicine & Rehabilitation; etc.), although other specialties and levels of training will be considered.

“Trainees in the Laboratory Track will have electives to choose from for the afternoon lab courses.

“The course is sponsored by Parent Project Muscular Dystrophy and the Muscular Dystrophy Association.

“Register at http://bit.ly/19MYO. In order to register for this course, the trainee must (1) fill out the form at the link, and (2) submit a CV or biosketch to kevin.flanigan@nationwidechildrens.org. I will confirm their eligibility for the course. Their registration will not be complete until they receive confirmation from me of their acceptance.”

Dr. Melissa Dixon at the University of Utah Department of Pediatrics is conducting this study. Dr. Dixon is a psychologist whose clinical interests include understanding cognitive function and the psychological and neurobehavioral symptoms associated with neuromuscular disorders in children and adults.

She is also interested in understanding psychological distress in children with chronic medical illness. Her research focuses on longitudinal assessment of cognitive function and brain connectivity and function in children with neuromuscular disease. (Get more information on Dr. Dixon’s credentials.)

Study Purpose

Cognitive Function and Neuroimaging in Myotonic Dystrophy (IRB_001116528)
The purpose of this study is to learn more about how congenital myotonic dystrophy and childhood-onset myotonic dystrophy affects thinking, memory, attention, brain function, and how these processes change over time. Dr. Dixon and her research team want to find out how the brains of children and adolescents with CDM and chDM1 are connected together through fibers that are like cable connections called white matter tracts, and how these connections may change with time, be related to age, and differ from the brains of children and adolescents without CDM or chDM1.

Study Design and Eligibility Criteria

Male and female children and adolescents (age 7 to 16 years) diagnosed with CDM or chDM1 are invited to participate. Participation includes two visits to the University of Utah. All study procedures will be conducted during a single-day outpatient visit at baseline (year 1) and 12 months (year 2). Study procedures include the consent/assent process, physical exam, completing questionnaires about how myotonic dystrophy affects behavior and quality of life, neuropsychological testing, and a MRI. The testing is approximately 4-6 hours per visit.

To Follow Up and Enroll

If you interested in participating or learning more about this study, please contact Melissa Dixon, PhD, MS, Department of Pediatrics, University of Utah, 15 North 2030 East, Room 2160A, Salt Lake City, Utah 84112. Dr. Dixon is also available by phone (office) 801-585-7606 or by email.

Speech disorders (dysarthria) in CDM and childhood-onset DM1 have long been recognized and surveillance by speech and language therapists is an important aspect of patient care. Facial weakness and myotonia, and involvement of oral cavity, palatopharyngeal and respiratory muscles, are known to contribute to speech impairment.

The Three Major Contributors to Speech Disorders 

In a recent review of speech disorders in DM1 (Lopes Cardos and Baptista, 2017), three major contributors to speech disorders were identified—myotonia as a hindrance to the initiation of speech, muscle weakness leading to reductions of lip force, and atrophy of tongue muscles. The authors note that few studies have evaluated the effectiveness of various speech therapies. There is no consensus on whether oral muscle exercises can improve lip strength. Other reports have shown that warming up reduced myotonia and led to increased speech rate and decreased variability in speech, but there were some concerns that this strategy could increase fatigue and thereby be counterproductive. Finally, increasing lip strength through exercising with an oral screen has been reported to increase lip force, but had no apparent effect on lip articulation. The authors concluded that strategies of warming up facial muscles and lip exercises can help, but used alone are insufficient to correct speech disorders in DM1 and therefore speech therapy is advised.

In this context, a new study has characterized the characteristics of speech in 50 subjects with CDM and childhood-onset DM1 (Sjőgreen et al., 2018). All subjects with CDM showed impairments of the intelligibility of their speech and nearly 80% of those with childhood DM1 were similarly impaired. The authors further characterized key speech components, identifying deficits in producing sounds: (1) that require coordinated function of both lips (bilabial consonants), (2) that require placing the tip of the tongue between the teeth (interdental consonants), and (3) that are due to increased airflow through the nose during speech (hypernasal speech). They also established a correlation between maximum lip force (as an indicator of how oral and facial muscles are affected in DM patients) and the intelligibility of speech. Some patients employed a variety of compensatory strategies to improve speech, including placing their tongue between their lips or biting the lower lip, to produce appropriate speech sounds—in some these strategies were very effective, but still did not reduce poor intelligibility in others.

The Connection Between DM and Speech Disorders

The researchers conclude that most children with CDM or childhood-onset DM1 will need speech therapy starting at a young age and that the most those with the severe manifestations will require training in alternative means of communication. Taken together, they show that weakness of oral and facial muscles is the primary cause of disordered speech in congenital and early-onset DM1. These findings suggest that therapies under development to improve muscle function in DM may also have positive effects on speech disorders. Finally, the research team reaffirmed conclusions of prior studies in that this patient group will require speech therapy from an early age.

References:

Myotonic dystrophy type 1 (DM1) and speech problems.
Lopes Cardoso I, Baptista H.
JSM Communication Dis. 1(1): 1003.
https://www.jscimedcentral.com/CommunicationDisorders/communicationdiso…

Speech characteristics in the congenital and childhood-onset forms of myotonic dystrophy type 1.
Sjögreen L, Mårtensson Å, Ekström AB.
Int J Lang Commun Disord. 2018 Jan 12. doi: 10.1111/1460-6984.12370. [Epub ahead of print]

Since a neuropsychological study in 2008 (Ekström et al., 2008), there have been few studies of pediatric cohorts to assess potential links between congenital and childhood-onset DM1 with autism spectrum disorders. The Ekström analysis of 57 children and adolescents with DM1 showed that 53% exhibited autism spectrum or other neuropsychiatric disorders (e.g., attention deficit hyperactivity disorder or Tourette's syndrome). The authors concluded that awareness of potential autism spectrum disorder comorbidity in DM1 was essential to patient care. There has been little literature on this issue since 2008.

A New Cohort Study of Autism and Childhood DM1

Dr. Nathalie Angeard (Paris Descartes University and Institut de Myologie) and colleagues recently published a review of nine studies focused on cognitive disorders in childhood DM1, compromising 175 cases (Angeard et al., 2017).

Emotional and behavioral disorders were prominent among reports in childhood DM1—the earlier study by Ekström and colleagues found that 36% of a cohort containing congenital (CDM) and juvenile-onset DM1 had autism spectrum disorders, although other studies did not report that high a prevalence. Angeard suggests that the association between and difficulties in the differential diagnoses of intellectual disability and autism spectrum might contribute to differences in reports of autism spectrum in CDM and juvenile DM1.

Cognitive function studies in CDM have reported moderate to severe intellectual disability in greater than half of patients studied. Considerable information is available regarding the characterization of specific cognitive function deficits and is reported in this meta-analysis. Patients with autism spectrum comorbidity did not fit a narrow profile, but rather exhibited a range of severity of symptoms, cognitive abilities and functional adaptations. The authors suggest that a considerable gap exists in understanding executive function and social cognition in childhood DM1, making it difficult to compare these patients with those with autism spectrum disorder. Likewise, a dearth of neuroanatomic and brain function studies in childhood DM1 also makes it difficult to compare their profile with that of autism spectrum disorder children. Where comparisons can be made based on available publications, the authors compare and contrast the social/communication, cognitive function and brain abnormality profiles between the two disorders (Table 2 in Angeard et al., 2017).

It’s Not Yet Clear Whether Childhood DM1 and Autism Spectrum Disorders are Comorbid

Overall, Angeard and colleagues note that only the Ekström paper reports high prevalence of autism spectrum disorder in childhood DM1 (36% versus 1% in the general population). Most publications agree, however, upon moderate prevalence of autism spectrum disorders in CDM. An evolving definition of autism spectrum over the time of the publications assessed here complicates any clear conclusion regarding comorbidity. The authors note that the prevalence of intellectual disability among childhood DM1 and autism spectrum may lead to biases in diagnosis. Taken together, they regard the question of comorbidity of childhood DM1 and autism as still open, requiring more careful cross-sectional and longitudinal natural history studies of the cognitive and behavioral phenotype of childhood DM1. For now, earlier attention to the cognitive, developmental, and social/emotional profiles of those at risk for CDM and juvenile-onset DM1 is warranted.

References:

Autism spectrum conditions in myotonic dystrophy type 1: a study on 57 individuals with congenital and childhood forms.
Ekström AB, Hakenäs-Plate L, Samuelsson L, Tulinius M, Wentz E.
Am J Med Genet B Neuropsychiatr Genet. 2008 Sep 5;147B(6):918-26. doi: 10.1002/ajmg.b.30698.

Childhood-onset form of myotonic dystrophy type 1 and autism spectrum disorder: Is there comorbidity?
Angeard N, Huerta E, Jacquette A, Cohen D, Xavier J, Gargiulo M, Servais L, Eymard B, Héron D.
Neuromuscul Disord. 2017 Dec 15. pii: S0960-8966(17)31337-8. doi: 10.1016/j.nmd.2017.12.006. [Epub ahead of print]

The Pathway to an Approvable (and Reimbursable) Therapeutic

DM offers many advantages in attracting drug developers, not the least of which is the ability to use changes in alternative splicing as a rapid and quantitative measure of target engagement/modulation and dosing. Beyond such early-stage milestones, a challenge in myotonic dystrophy is finding endpoint measures that: (a) are clinically meaningful to patients and caregivers, (b) show progressive change during the timeframe of a typical clinical trial (6-12 months) and (c) provide sufficient validity, responsiveness and reproducibility as to support acceptance by both regulators and payers.

An insightful pair of review articles in the NEJM (Pocock and Stone, 2016a, 2016b) discuss interpreting data from clinical trials, pointing out the importance of selection of outcome measures and their underlying methodology: “Trial success may hinge on definitions of the outcomes and on the methods used for their adjudication.” How do we get there for DM?

There is no easy pathway toward adequate, clinically meaningful outcome measures, particularly in a slowly-progressive, multi-system disorder like DM. The necessary ‘grunt work’ involves triaging and implementing a broad battery of putative outcome measures and applying them in sufficiently powered, longitudinal natural history studies. To avoid siloing of small, underpowered datasets, natural history studies in DM should plan, from the beginning, that data sharing will occur in order to achieve broader goals that are un-addressable any other way (Larkindale and Porter, 2017).

‘Responsive’ Outcome Measures for DM1

Drs. Marie Kierkegaard (Karolinska Institute), Cynthia Gagnon (University of Sherbrooke) and colleagues have published the results of a 9-year, longitudinal natural history study assessing function, disabilities and overall health in a cohort of subjects with adult- and late-onset DM1. The responsiveness of a wide battery of functional tests and patient-reported perceptions of changes in relevant functions was evaluated at baseline and 9 years later in 113 subjects seen at the Saguenay Neuromuscular Clinic.

Patient reports of perceived change in specific functions (balance, walking, lower-limb weakness, stair-climbing and hand weakness) correlated with those changes measured by the research team. The highest degree of responsiveness was found for measures of mobility, balance and muscle strength (Timed Up and Go, Berg Balance Scale and Quantitative Muscle Testing). By contrast, outcome measure responsiveness was poorer for manual dexterity and grip strength.

Significantly, some measures -- Timed Up and Go, grip strength, pinch-grip strength and Purdue Pegboard Test -- did not show longitudinal changes that exceeded known measurement errors, raising questions about value for clinical trials.

Criticality of Endpoint Selection for Clinical Trials

Taken together, this study provides important insights into the responsiveness of a range of functionally significant outcome measures that could be selected for interventional clinical trials. Assessment of endpoint measure change was made over a time period considerably longer than any feasible clinical trial (9 years), and DM1-related cognitive decline over the study period likely influenced patient perception abilities, but these data help improve understanding of the relative performance of specific measures. Some measures did not distinguish either clinically important changes from known measurement errors and/or the smallest detectable change for the specific instrument. These findings should be considered, along with data from other studies, when defining suitable outcomes and measurement methodology for DM1 interventional clinical trials. As the authors note, it is essential to assess outcome measure responsiveness over the shorter time period that is feasible for clinical trials.

References:

Responsiveness of performance-based outcome measures for mobility, balance, muscle strength and manual dexterity in adults with myotonic dystrophy type 1.
Kierkegaard M, Petitclerc É, Hébert LJ, Mathieu J, Gagnon C.
J Rehabil Med. 2017 Dec 20. doi: 10.2340/16501977-2304. [Epub ahead of print]

The Primary Outcome Fails - What Next?
Pocock SJ, Stone GW.
N Engl J Med. 2016 Sep 1;375(9):861-70. doi: 10.1056/NEJMra1510064

The Primary Outcome Is Positive - Is That Good Enough?
Pocock SJ, Stone GW.
N Engl J Med. 2016 Sep 8;375(10):971-9. doi: 10.1056/NEJMra1601511.

Seeking a better landscape for therapy development in neuromuscular disorders.
Larkindale J, Porter JD.
Muscle Nerve. 2018 Jan;57(1):16-19. doi: 10.1002/mus.25961. Epub 2017 Sep 23.

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 https://clinicaltrials.gov/ct2/show/NCT02858908).

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.

References:

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.

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