Research Posters - 2022 MDF Annual Conference

Traditional poster sessions combine a visual representation of the findings of a paper/study – the poster - with the opportunity for individualized, informal discussion of the presenter’s work. MDF is thrilled to make this opportunity available at this year’s conference in a virtual format, in which special Web rooms will be designed for attendees to view posters. Click here to learn more about the 2022 MDF Annual Conference.

Use the table of contents below to quickly jump to a specific abstract:

1. In vitro reduction of RNA foci and restoration of alternative splicing in myotonic dystrophy type 1 using AAV8.U7-snRNAs.
2. Myotonic dystrophy, surgeries, cardiovascular testing, mobility aids and healthcare utilization based on a 5-year real-world data analysis.
3. Lead antimiR-23b drug rescues cell phenotypes in a range of Myotonic Dystrophy genetic backgrounds.
4. A reductionist approach studying the outcome of altered splicing in Myotonic Dystrophy.
5. Electrical Impedance Myography Predicts Muscle Function in DM1 Patients.
6. Mitochondrial dysfunction in Myotonic Dystrophy type 1 patients.
7. Evaluation of Cognitive Function, Cerebral Structure and Functional Connectivity in Children with Congenital Myotonic Dystrophy and Childhood-Onset Myotonic Dystrophy Type 1.
8. DYNE-101 Targets DMPK Expression to Correct Splicing in Key Muscles for DM1 Pathology and Is Well Tolerated in Cynomolgus Monkeys.
9. Enhanced Anesthetic, Benzodiazepine Sensitivity, and GABAAR mRNA Dysregulation in a MBNL2 Knockout Mouse Model of Myotonic Dystrophy.
10. Generation and Characterization of a DM2 BAC Mouse Model.
11. Assessing therapeutic potential and mechanism of action of novel small molecules in Myotonic Dystrophy type 1.
12. A cognitive portrait of adults living with the DM1 childhood phenotype.
13. Developing an RNA-targeted therapeutic for microsatellite repeat expansion disorders using catalytically dead Cas13d.
14. RNA subcellular mislocalization in myotonic dystrophy type 1 (DM1) patient iPSC-derived neurons.
15. Genetic characterization of expanded CTG repeats in new DM1 knockin mouse models.
16. Initial Psychometric Properties of the Congenital Myotonic Dystrophy Type 1 Rating Scale (CDM1-RS).
17. AAV9-mediated overexpression of MBNL rescues DM1 cardiac phenotypes.
18. Evaluation of in vitro gene editing removal of repeat expansion as potential treatment for myotonic dystrophy type 1.
19. Microscopy-Based Identification of RNA Foci Modulators in Myotonic Dystrophy Type 1.
20. Choroid plexus spliceopathy in myotonic dystrophy type 1.
21. A Novel EEV-Conjugated PMO, ENTR-701, Reduces Nuclear Foci and Corrects Aberrant Splicing in Myotonic Dystrophy Type 1 Preclinical Models.
22. Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy.
23. Identifying Novel Metabolic Targets for Myotonic Dystrophy type 1.
24. A CTG repeat-selective screen of a natural product library reveals dietary natural compounds as potential therapeutics for myotonic dystrophy.
25. Specialists involved in the diagnosis and management of myotonic dystrophy based on a real-world data analysis pre-and post-diagnosis.
26. Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function.
27. TREAT-NMD Myotonic Dystrophy Global Registry Network: An International Collaboration in Myotonic Dystrophy Type 2.
28. Injury Among Adults with Myotonic Dystrophy Type 1: Findings from Linkage of South Carolina and Utah’s Muscular Dystrophy Surveillance, Tracking, and Research Network Data to Statewide Emergency Department Encounters.
29. TREAT-NMD Myotonic Dystrophy (DM) Global Registry Network: An update in 2022.
30. Circadian rhythm disruptions in Myotonic Dystrophy Type 1 (DM1) – a multi-systemic genetic disease with major CNS involvement.
31. Single-administration of a cyclic peptide-conjugated CUG-repeat steric blocker rescues myotonia and molecular phenotypes in HSA^LR mice.
32. Elimination of myotonia from mouse models of myotonic dystrophy type 1.
33. Analysis of DMPK expansion transcript degradation in DM1.
34. Building connections to reduce barriers to therapies.
35. Myotonic Dystrophy Family Registry.
36. The Global Alliance for Myotonic Dystrophy Awareness efforts to launch International Myotonic Dystrophy Awareness Day.

Research Poster Presentation Abstracts

Poster abstracts are listed in alphabetical order by corresponding author. An * denotes a current MDF Research Fellow.

1. In vitro reduction of RNA foci and restoration of alternative splicing in myotonic dystrophy type 1 using AAV8.U7-snRNAs

   Camila F. Almeida¹, Ding Li¹, Robert B. Weiss², and Nicolas Wein^{1,3 
   1}Center for Gene Therapy, Nationwide Children’s Hospital, Columbus, Ohio, USA, ²Department of Human Genetics, The University of Utah School of Medicine, Salt Lake City, Utah, USA, ³Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA.

   Myotonic dystrophy type 1 is the most common form of muscular dystrophy in adults affecting the skeletal muscle, heart, and brain. It is caused by a CTG repeat expansion in the 3’UTR of the DMPK gene. Antisense oligonucleotides aiming to knock down DMPK expression or bind to the CTGs have been explored but require repeated injections to sustain therapeutic effects. We designed modified U7 small nuclear RNAs containing a promoter and antisense sequences targeting DMPK to overcome this limitation. The AAV8.U7snRNAs (produced by Audentes/Astellas Pharma) target two exons to induce an out-of-frame skipping, resulting in transcript degradation, and additional antisense sequences target the 3’UTR region to promote steric hindrance. We derived and characterized FibroMyoD cell lines from DM1 skin fibroblasts. FibroMyoD cells were transdifferentiated into myotubes which were transduced with the AAV8.U7snRNAs. The effect of the treatment was evaluated using RNA FISH combined with MBNL1 immunostaining for foci quantification and MBNL1 localization. Gene expression and splicing profiles were assessed by RNAseq. Selected candidate genes were confirmed by RT-PCR. The FibroMyoD cells reproduced the main DM1 molecular hallmarks, proving to be suitable for in vitro screening of gene therapies. The AAV treatment reduced the foci number, changed MBNL1 localization, and shifted the splicing profile. Vectors targeting the 3’UTR region were the most effective. In conclusion, DM1-FibroMyoD cells are an important source to investigate the disease pathology and testing potential therapies. AAV8.U7snRNAs constructs reversed the DM1 pathology in vitro, encouraging further investigation on the use of these vectors to treat DM1.

2. Myotonic dystrophy, surgeries, cardiovascular testing, mobility aids and healthcare utilization based on a 5-year real-world data analysis

   John W. Day¹, Richard A. Brook³, Kathryn A. Munoz², Nathan L. Kleinman³, Chao-Yin Chen², Brad McEvoy², Kelly DiTrapani², Mark Stahl², and Li-Jung Tai^{2
   1}Stanford Neuroscience Health Center, Stanford, California, USA; ²Avidity Biosciences, Inc., San Diego, California, USA; ³Better Health Worldwide, Newfoundland, New Jersey, USA

   Objective: Describe the management of myotonic dystrophy (DM) patients compared with matched controls (MCs) in the five years following diagnosis. Design/Methods: We used PharMetrics deidentified US claims (Jan-2010—Mar-2021) to retrospectively evaluate care for DM-patients vs non-DM MCs. DM patients were required to have ≥2 DM claims ≥30 days apart (index date=first diagnosis date). Three non-DM controls were matched to each DM-patient. All subjects had five years of data following their index date. All cost data were adjusted to constant 2020 dollars. Data reported are per-member-per-year for cost, number of services and days of service. All reported findings significant (p<0.05). Results: We identified 892 DM-patients and 2676 MCs. In the 5 years following diagnosis, across all locations of care, DM-patients had higher mean: costs [$21,728 (s.d.$50,684)-vs-$6,221 ($13,347) for MCs], more services [93.40 (118.53)-vs-42.61 (52.68)], and more days of care [35.77 (39.31)-vs-16.76 (18.90)]. DM-patients were more likely to: use walking aids or wheelchairs (21.1%-vs-4.6%), have an electrocardiogram (ECG 85.1%-vs-39.0%) and echocardiogram (65.0%-vs-15.1%). DM-patients underwent more surgeries for: cardiovascular (85.7%-vs-67.6%), eye (23.0%-vs-6.6%), gastrointestinal (43.0%-vs-27.1%), auditory (15.6-vs-6.1%), musculoskeletal (33.5%-vs-25.9%), urinary (12.4%-vs-6.9%), respiratory (11.7%-vs-6.4%), and nervous systems (15.0%-vs-10.5%). Conclusions: Healthcare utilization was significantly higher in DM-patients following diagnosis compared with controls. With the lack of an approved therapy, the increased utilization likely reflects manifestations of managing DM. The data reflect the financial burden on DM-patients and their families. Since there are no disease-modifying therapies for DM, new therapies are needed to address the high unmet need.

3. Lead antimiR-23b drug rescues cell phenotypes in a range of Myotonic Dystrophy genetic backgrounds.

   Estefanía Cerro-Herreros^1,2,3, Judit Núñez-Manchón⁴, Neia Naldaiz-Gastesi^5,6, Marc Carrascosa-Sàez³, Andrea García-Rey^1,2, Irene González-Martínez^1,2, Kevin Moreno³, Javier Poyatos-García^7,8,9, Juan J Vilchez^7,8,9, Adolfo López de Munain^5,6,10,11, Mònica Suelves⁴, Gisela Nogales-Gadea^4,9, Beatriz Llamusí³ and Rubén Artero^{1,2
   1}University Research Institute for Biotechnology and Biomedicine (BIOTECMED), Universidad de Valencia, Valencia, Spain; ²Translational Genomics Group, INCLIVA Biomedical Research Institute, Valencia, Spain. Avenue Menéndez Pelayo 4 acc, 46010, Valencia, Spain; ³ARTHEx Biotech. Parque Científico de la Universidad de Valencia. Calle del Catedrático Agustín Escardino Benlloch, 9, 46980 Paterna, Valencia; ⁴Neuromuscular and Neuropediatric Research Group, Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP), Campus Can Ruti, Universitat Autònoma de Barcelona, 08916 Badalona, Spain; ⁵Neuromuscular Diseases Group, Neurosciences Area, Biodonostia Health Research Institute, 20014 Donostia/San Sebastián, Spain; ⁶CIBERNED, Carlos III Institute, Spanish Ministry of Economy & Competitiveness, 28031 Madrid, Spain; ⁷The IISLAFE Health Research Institute, Avenida Fernando Abril Martorell, 106 Torre A 7 planta, 46026 Valencia, Spain; ⁸Neuromuscular Reference Centre ERN EURO-NMD and Neuromuscular Pathology and Ataxia Research Group, Hospital La Fe Health Research Institute, Valencia, Spain; ⁹Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain; ¹⁰Department of Neurology, Donostialdea Integrated Health Organization, Osakidetza Basque Health Service, 20014 Donostia/San Sebastián, Spain; 11Department of Neurosciences, Faculty of Medicine and Nursery, University of the Basque Country UPV-EHU, 20014 Donostia/San Sebastián, Spain.

   Background: Symptom heterogeneity is characteristic of Myotonic Dystrophy type 1 (DM1) and stems, at least in part, from the somatic instability of the CTG repeat expansions, which vary from one tissue to another and during aging. Thus, experimental therapies must work under various repeat expansion size conditions in patients. Objective and Methodology: To assess the therapeutic potential of antimiR-23b, an antisense oligonucleotide blocker of MBNL1 repressor miR-23b, we treated eight independent human DM1 primary myoblast lines. Myoblasts ranged from 117 to 1054 CTG repeat sizes and included males and females aged 36 to 49 years at the time of biopsy. The therapeutic potential was assessed by analyzing DM1 cell phenotypes, including fusion index, foci number and MBNL1 aggregates, miss-splicing, and expression levels of target miRNA, DMPK mRNA, and MBNL1 protein. Results: miR-23b was found to be upregulated in patient myoblasts, and reduction in miR-23b activity in all cases increased MBNL1 protein and reduced DMPK expression and foci in treated cells after ten days. Furthermore, the combined increase in the total amounts of MBNL1 protein and reduction in mutant DMPK-mediated sequestration of MBNL1 produced a significant rescue of MBNL1-dependent splicing events and differentiation capacity, measured as an increase in the fusion index and myotube area. Data analysis also suggests a correlation between CTG expansion size and miR-23b overexpression, and between the degree of MBNL1 enhancement and splicing rescue. Conclusion: We conclude that antimiR-23b oligonucleotides currently being developed (Arthex Biotech’s X82108/ATX-01), have therapeutic potential across very different genetic backgrounds.

4. A reductionist approach studying the outcome of altered splicing in myotonic dystrophy

   Lily Cisco*¹, Matthew Sipple¹, Katherine Edwards¹, Sakura Hamazaki², and John Lueck^{1,3
   1}University of Rochester, Department of Pharmacology and Physiology, New York, USA; ²University of Rochester, Department of Biology, New York, USA; ³University of Rochester, Department of Neurology, New York, USA.

   Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. In DM1, there are hundreds of aberrantly spliced transcripts, making it challenging to connect splice variants to disease manifestations. The mechanism behind muscle weakness and atrophy remains poorly understood, but a correlation between altered splicing in transcripts central to excitation-contraction coupling and muscle weakness in DM1 patients has been made. These alternatively spliced transcripts encode the skeletal muscle L-type voltage gated calcium channel (Ca_v1.1), the sarcoplasmic calcium release channel (RyR1), and the sarcoplasmic reticulum calcium pump (SERCA1). We hypothesize that calcium homeostasis in skeletal muscle is altered with these splice variants leading to calcium toxicity and muscle degeneration. We have generated mouse models that express DM1 isoforms of Ca_v1.1, RyR1, and SERCA1. Further, we have also bred these models together to generate double- and triple-trouble mice. We will use these models to understand the effects of aberrantly spliced Ca_v1.1, RyR1, and SERCA1 individually on skeletal muscle without the complex alternative splice background, as well as determine if there is a phenotypic tipping point when combined with one another. To date, we have found that survival is unaffected in all our mouse models, but all mice exhibit increased fatigue during an exhaustive treadmill protocol. We will further our study with in vitro muscle contraction, histology, fiber-typing, and voluntary wheel running to understand the impacts of these alternative splice variants alone and in combination.

5. Electrical impedance myography predicts muscle function in DM1 patients

   Parker Conquest, Brigham McKee, Elizabeth Cornforth, Alex Sizemore, Elise Townsend, Seward Rutkove, and Thurman Wheeler
   Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA.

   Objective: To evaluate electrical impedance myography (EIM) as a non-invasive indicator of muscle disease status in DM1. Methodology: Using the Myolex mScan, we applied alternating current at 41 frequencies (1 - 10,000 kHz) and measured the resulting voltage in 7 muscle groups of DM1 (N = 21) and unaffected (N = 8) subjects. From the resistance and reactance, we calculated the phase angle—the time shift of electric current as it passes through muscle. All subjects also underwent quantitative muscle function testing. A subset of 14 DM1 subjects were re-evaluated at 6-to-8 months. Results: Mean phase angle at 100 kHz is significantly reduced in the wrist flexor, wrist extensor, gastrocnemius, and tibialis anterior (TA) muscles of DM1 vs unaffected subjects. Phase angle correlates with strength in the biceps, triceps, wrist flexor, wrist extensor, gastrocnemius, and TA (r 0.62 - 0.78; P < 0.0001). TA and gastrocnemius phase angles correlate with 6-minute-walk distance (r 0.58 and 0.59; P 0.01 and 0.009) and 10-meter-walk time (r -0.60 and -0.52; P 0.01 and 0.02). Phase angle values are reproducible from side-to-side and between first and second visits. Conclusions: EIM phase angle is a candidate biomarker of disease status in DM1 muscle tissue. Funding: Elaine and Richard Slye Fund; U.S. Department of Defense; U.S. National Institutes of Health.

6. Mitochondrial dysfunction in myotonic dystrophy type 1 patients

   Valeria Di Leo^1,2, C Lawless^1,2, M-P Roussel³, GS Gorman¹, OM Russell^1,2, HA Tuppen¹, E Duchesne⁴, and AE Vincent^{1,2
   1}Wellcome Centre for Mitochondrial Research, Newcastle University, Newcastle upon Tyne, UK; ²Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK; ³Department of Fundamental Sciences, University of Québec in Chicoutimi, Québec, Canada; ⁴Department of Health Sciences, University of Québec in Chicoutimi, Québec, Canada.

   Myotonic dystrophy type 1 (DM1) is an inherited type of muscular dystrophy, for which no viable treatment exists yet. Resistance exercise intervention has been investigated as a potential way to improve skeletal muscle (SKM) weakness in DM1 patients. The aim of this study is to investigate the oxidative phosphorylation (OXPHOS) in SKM biopsies from a cohort of DM1 patients (n=10 males), who previously undertook a 12-week resistance exercise training. Immunofluorescence labelling was used to investigate Laminin, VDAC1 (mitochondrial mass), Ndufb8 (Complex I - CI - subunit), and COX1 (Complex IV - CIV - subunit). Using a linear model between VDAC1 and Ndufb8 or COX1, the 95% predictive intervals for the fibers of the combined controls population were used to classify patient fibers as being normal, deficient/low (^low) level or high (^high) level. Before exercise, all patients, except P01, present with CI deficiency, and some show additional CIV deficiency. After exercise, seven patients out of 10 display a significant reduction in Ndufb8^low fibers (p < 0.03), and the majority of patients present a significant reduction in COX1^low fibers (p < 0.02) Furthermore, patients who do not show ameliorations in either CI or CIV deficiency, display a significant increase in COX1^high fibers (p < 0.001). Additionally, four patients have a significant increase in mitochondrial mass after exercise (p < 0.0001). For the first time, we demonstrate that DM1 patients may present with mitochondrial dysfunction in both CI and CIV in SKM tissue. Resistance exercise training rescues the OXPHOS defects both decreasing the deficiency and increasing CI and CIV protein levels.

7. Evaluation of cognitive function, cerebral structure and functional connectivity in children with congenital myotonic dystrophy and childhood-onset myotonic dystrophy type 1

   Melissa M. Dixon¹, Jadwiga Rogowska², Punitha Subramaniam², Brith Otterud¹, Lauren Branigan¹, Rebecca Crockett¹, Emily Woolsey¹, Nicholas Johnson³, Russell J. Butterfield¹, and Deborah Yurgelun-Todd^{2
   1} Department of Pediatrics, University of Utah, USA; ²Department of Psychiatry, University of Utah, USA; ³ Department of Neurology, Virginia Commonwealth University, USA

   Introduction: Cognitive function is significantly impaired in children with congenital myotonic dystrophy (CDM) and childhood-onset myotonic dystrophy, type 1 (chDM1). Objective: Identify key cerebral structural and functional connectivity factors predictive of cognitive function deficits in CDM/chDM1. Methods: Thirty participants with CDM/chDM1 and controls completed neuropsychological, structural and resting state functional magnetic resonance imaging (rsfMRI) evaluations. A 3T scanner, DPARSFA toolbox and SPM8 were used for image collection and processing. Group differences in brain structure and functional connectivity were examined. Regression analysis was used to identify relationships between neuropsychological measures and neuroimaging data. Results: Preliminary data revealed significant differences in brain structure and functional connectivity in CDM/chDM1; greater connectivity between the cingulate cortex and prefrontal brain regions in CDM; strong associations between cognitive function and cingulate cortex – prefrontal brain regions connectivity in CDM. Conclusion: Our results are consistent with previous reports of prefrontal white matter changes in myotonic dystrophy type 1, and suggest that anomalous prefrontal development plays a key role in the cognitive function deficits observed in children with CDM and chDM1.

8. DYNE-101 targets DMPK expression to correct splicing in key muscles for DM1 pathology and is well tolerated in cynomolgus monkeys

   Stefano Zanotti, Tyler Picariello, Lydia Schlaefke, Ryan Russo, Ann Chang, Scott Hildebrand, John Najim, Qifeng Qiu, Timothy Weeden, John W Davis II, Ash Dugar, and Oxana Beskrovnaya
   Dyne Therapeutics, Inc., Waltham, Massachusetts, USA.

   Objective: The FORCE^TM platform was designed to overcome the limitations in the development of therapeutics for muscle diseases and consists of an antigen-binding fragment-antisense oligonucleotide (Fab-ASO) conjugate that targets the human transferrin receptor (TfR)1 to enhance ASO uptake into muscle. We are developing DYNE-101, a Fab conjugated to an ASO that targets the human DMPK RNA, for the treatment of DM1. Methodology: To determine the potential of DYNE-101 to drive disease modification, a novel hTfR1/DMSXL mouse expressing human TfR1 and a human DMPK with >1,000 CUG repeats was generated. Results: In hTfR1/DMSXL mice, DYNE-101 led to a 40-49% reduction of toxic human DMPK RNA and a corresponding splicing correction in cardiac and skeletal muscle that were accompanied by a 49% reduction of DMPK nuclear foci in the heart. Of note, monthly dosing of DYNE-101 in hTfR1/DMSXL mice resulted in a robust, dose-dependent, reduction of mutant human DMPK RNA in muscle. To establish the translatability of these observations to primates, male cynomolgus monkeys were subjected to 2 monthly doses of DYNE-101. This treatment led to a substantial and dose-dependent suppression of WT DMPK expression up to 70% in cardiac, skeletal, and smooth muscle. Data in mice and monkeys reveal that DYNE-101 displays strong pharmacological activity with low and infrequent administration. Importantly, DYNE-101 was well-tolerated after repeated administration in a 13-week GLP toxicology study in cynomolgus monkeys. Conclusions: These data demonstrate that DYNE-101 effectively reduces DMPK expression in multiple muscles affected by DM1 pathology and support advancement of DYNE-101 into the clinic.

9. Enhanced anesthetic, benzodiazepine sensitivity, and GABAAR mRNA dysregulation in a MBNL2 knockout mouse model of myotonic dystrophy

   Kamyra S. Edokpolor*, Zachary T. McEachin, Anwesha Banerjee, Eric T. Wang, Paul S Garcia, and Gary J. Bassell
   Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, USA; Department of Molecular Genetics & Microbiology, Center for Neurogenetics, University of Florida-Gainesville, Gainesville, Florida, USA; Department of Anesthesiology, Columbia University, New York, New York, USA.

   Molecular mechanisms underlying increased risk of surgical complications associated with general anesthesia in individuals with DM1 are not well understood. Some general anesthetics used in the clinic exert their inhibitory effects by dynamically regulating the activities or levels of GABA_A extra-synaptic receptors (GABA_AR.) Loss of Muscle blind-like protein 2 (MBNL2) in mice results in CNS symptoms that phenocopy DM1, as well as various pre-mRNA mis-splicing events, such as increased production of Gabrg2 mRNA encoding the 2S isoform of the γ2 subunit of GABA_A receptors. Gamma 2S-containing GABA_ARs are enriched at extra-synaptic sites, exhibit enhanced GABA transmission and reduced desensitization and are more sensitive to benzodiazepines (positive allosteric GABA_AR modulators, PAM). Thus, we hypothesized that alterations in GABA_AR subunit expression and sensitivity may underlie anesthesia-related CNS symptoms in DM1. Here, we validate via rt-qPCR that γ2S vs. γ2L ratios are elevated in Mbnl2 KO brain. We further test the hypothesis that MBNL2 depletion leads to enhanced GABA sensitivity using behavioral paradigms. Sevoflurane, an anesthetic predominantly mediated by GABA_AR, as well as diazepam (PAM) and Zolpidem, agonists that target the benzodiazepine site of GABA_AR, were administered to Mbnl2 KO mice. Delayed recovery following sevoflurane were observed in Mbnl2 KO mice compared to their wild type littermates. Mbnl2 KO mice also displayed longer sedation periods after Zolpidem administration. Taken together, our findings suggest that loss of MBNL2 function in DM1 may lead to altered GABAergic sensitivity, with implications for neurotransmission in myotonic dystrophy.

10. Generation and characterization of a DM2 BAC mouse model

    Avery Engelbrecht*^1,2, Lisa E.L. Romano^1,2, Kiruphagaran Thangaraju^1,2, S. Elaine Ames^1,2, John Cleary^1,2, Tao Zu^1,2, and Laura P.W. Ranum^{1,2
    1}Center for NeuroGenetics, ²Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA.

    Objective: Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic diseases caused by CTG or CCTG repeat expansions located in the DMPK or CNBP genes, respectively. RNA gain of function effects, bidirectional transcription and repeat associated non-ATG (RAN) translation are all found in DM. RAN translation of sense (CCUG) and antisense (CAGG) expansion transcripts produce (LPAC) and (QAGR) RAN proteins. LPAC and QAGR proteins are toxic to cells and found in brain regions with neurodegenerative changes and white matter loss. Understanding the role of RAN proteins in DM2 and developing therapeutic approaches requires animal models that mirror DM2 patient disease features. Methodology: Using a bacterial artificial chromosome (BAC) approach we have generated two separate lines of DM2 BAC transgenic mice. We are currently characterizing these mice for RNA foci using HCR FISH, repeat instability by long-range PCR, histopathological and behavioral features. Results: Both DM2 mouse lines contain the entire CNBP gene with substantial flanking sequence and unstable expanded repeats ranging in size from ~750-1300 CCTGs. Southern blot analyses suggest a single insertion site in each line. HCR-FISH detects signal in transgenic mice for CCUG repeats in skeletal muscle and brain. Conclusions: We have generated a novel DM2 BAC transgenic mouse model that shows initial molecular phenotypes. We are continuing to characterize these mice and hope that this model will provide a useful tool for better understanding the molecular mechanisms of DM2 and therapy development.

11. Assessing therapeutic potential and mechanism of action of novel small molecules in myotonic dystrophy type 1

    Jesus A Frias*^1,2, H Mazdiyasni², JA Berglund^{1,2
    1}Department of Biological Sciences and ² The RNA Institute, State University of New York at Albany, New York, USA.

    Objective: Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by a CTG repeat expansions in the 3’ UTR of the DMPK gene. The resulting toxic expansion RNAs sequester the muscleblind-like (MBNL) family of alternative splicing regulators, leading to their loss of function and alternative splicing dysregulation which are connected to many DM1 symptoms (reviewed by Reddy et al., 2019). Small molecules, such as diamidines, have been used to therapeutically target the effect of toxic RNAs (Siboni et al., 2015) but these compounds can be toxic, have off-target effects, and/or display modest splicing rescue (Jenquin et al.,2018). To address these issues, the Berglund lab has designed and synthesized a series of novel small molecules, called modified polycyclic compounds (MPCs). Our object is to test the therapeutic potential and mechanism of action of these MPC for the treatment of DM1. Methodology: We treated DM1 patient-derived fibroblasts and myotubes with MPCs, extracted RNA and analyzed changes in alternative splicing events and expression of DMPK and MBNL transcripts via RT-qPCR. We also conducted MTT assays to assess the cell viability following MPC treatment. Results: Our preliminary data shows that multiple MPCs rescued mis-splicing in the 8-16nM (fibroblasts) or 62.5-125nM (myotubes) range, with no significant effects on cell viability. Treatment with MPCs also upregulated MBNL2 transcript levels in both cell types suggesting a potential mechanism of action (MOA). Conclusions: Our novel MPCs rescued mis-splicing at nanomolar levels, offering improved efficacy over previous diamidine compounds. While upregulation of MBNL is a potential MOA, further works is required to fully understand and optimize the therapeutic potential of MPCs.

12. A cognitive portrait of adults living with the DM1 childhood phenotype

    Benjamin Gallais^1,2, C Gagnon^2,3, A Magot⁴, R Chasserieau⁴, J Dolbec², S Muslemani^2,3, and L Richer^{2,5
    1}ÉCOBES – Recherche et transfert, Cégep de Jonquière, Jonquière, Qc, Canada; ²Groupe de recherche interdisciplinaire sur les maladies neuromusculaires (GRIMN), Jonquière, Qc, Canada; ³Université de Sherbrooke, Sherbrooke, Qc, Canada; ⁴Centre de référence Neuromusculaire AOC, CHU de Nantes, Nantes, France; ⁵Université du Québec à Chicoutimi, Chicoutimi, Qc, Canada.

    Objective: Myotonic dystrophy type 1 (DM1) is an autosomal dominant inherited disorder. As a multisystemic disease, it affects several systems, including the central nervous system. Children with the childhood phenotype usually presents lower IQ levels and cognitive impairments. However, only case reports have addressed cognitive impairments among adults with this phenotype. The aim of the present study is to assess an intellectual, cognitive and social cognition portrait in adults with the DM1 childhood phenotype. Methods: Fifty adult patients (M:25; F:25; 19-57yo) from Saguenay (Canada; n:35) and Nantes (France; n:15) regions were assessed with a neuropsychological battery, including intellectual, visuospatial, visuo-constructive, organisational, and executive functions, as well as emotion recognition and theory of mind. The battery combined classic paper-pencil, as well as computerized tests. Results: The evaluation revealed a mean IQ of 76, which correspond to a borderline qualitative classification. Moreover, significant deficits were observed on all cognitive and social cognitive assessed abilities. The proportion of participants below 1 SD ranged from 58 to 90%, depending on the subtests. Conclusions: Adults living with the DM1 childhood phenotype show intellectual and cognitive impairments that may affect their daily-life activities quality and achievements. These results highlight the relevant need for early academic and rehabilitation services, and the importance to aware and empower caregivers about cognitive and social difficulties; these abilities being crucial for daily-life autonomy and social participation.

13. Developing an RNA-targeted therapeutic for microsatellite repeat expansion disorders using catalytically dead Cas13d

    Valerie Gonzalez*, Allison Li* Maya Gosztyla, Suprita Mantravadi, Samantha Sison, and Gene Yeo
    Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA; * indicates co-first authors.

    Myotonic dystrophy type 1 (DM1) is one of many diseases caused by microsatellite repeat expansions (MREs). Knocking down the MRE-containing RNAs is an attractive therapeutic strategy for treating these conditions. However, previous work has largely relied on Cas13d, a protein with questionable therapeutic utility due to its high levels of off-target RNA cleavage. In an effort to design a robust system for knocking down MRE-containing RNA, we have utilized PIN endonuclease fused to catalytically dead Cas13d (dCas13d). Our system takes advantage of Cas13d’s RNA-targeting capabilities while avoiding the unwanted off-targeted effects that have been previously associated with its native endonuclease domain. To test our system, we transfected human embryonic kidney (HEK293T) cells with three plasmids containing 1) PIN-dCas13d, 2) a guide RNA (gRNA) targeting CAG repeats, and 3) exon 1 of the huntingtin (Htt) gene containing 74 CAG repeats. We then quantified the CAG repeat RNA using RNA dot blots. We saw that both fusion orders (N-terminal or C-terminal PIN) resulted in strong knockdown of CAG repeat RNA when compared to a non-targeting gRNA. Additionally, we observed a significant reduction in repeat-expanded Htt protein expression with our system. We are now testing our system’s efficacy in HEK293T cells expressing 105 CUG repeats, the MRE implicated in DM1. We are also testing both CAG- and CUG-targeting dCas13d-PIN as adeno-associated virus therapeutics in brain organoid models of DM1 and Huntington’s disease to examine on- and off-target effects of our system on the transcriptome. Our preliminary results demonstrate that PIN-dCas13d could be an effective research tool and/or therapeutic system for knocking down MRE-containing transcripts.

14. RNA subcellular mislocalization in myotonic dystrophy type 1 (DM1) patient iPSC-derived neurons

    Maya L Gosztyla*¹, Allison Li¹, Kathryn H Morelli¹, and Gene W Yeo^{1
    1}Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, USA.

    Myotonic dystrophy type 1 (DM1) is primarily characterized as a disease of abnormal splicing, which gives rise to progressive myopathy and myotonia. However, DM1’s cognitive symptoms do not appear to be fully explained by missplicing of neuronal transcripts. We hypothesized that RNA mislocalization contributes to neuronal phenotypes in DM1. We differentiated neurons from DM1 patient-derived iPSCs and neurotypical controls. Then we utilized a version of subcellular fractionation modified for neuronal cells, followed by RNA sequencing, to characterize RNA subcellular localization. When comparing DM1 to control neurons, we observed hundreds of mislocalized transcripts, most of which became more cytosolic in DM1. These changes could not be fully explained by changes in overall gene expression. We observed that GC content is positively correlated with more nuclear enrichment in DM1. Nuclear-shifted transcripts included many genes important for muscle and/or cardiac development, as well as components of the extracellular matrix, a result that is consistent with DM1 phenotypes. In contrast, cytosolic-shifted transcripts were highly enriched for the “plasma membrane” GO cellular component term and included multiple families of cell surface receptors. Many of these are G protein-coupled receptors, whose translation and transport to the plasma membrane are known to be highly dependent on specific RNA localization, suggesting these changes may have functional consequences at the protein level. Furthermore, cell fractionation of iPSCs revealed distinct patterns of mislocalization compared to neurons, suggesting that these changes derive from a neuron-specific mechanism. Our results highlight neuronal RNA subcellular localization defects as an important layer of dysregulation in DM1.

15. Genetic characterization of expanded CTG repeats in new DM1 knockin mouse models

    Christina S. Heil*¹, Zhenzhi Tang², and Charles A. Thornton^{2
    1}Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, USA; ²Department of Neurology, University of Rochester Medical Center, Rochester, New York, USA.

    Instability of expanded CTG repeats in the 3’-untranslated region (3’UTR) of the myotonic dystrophy protein kinase (DMPK) gene is a key driver for disease progression in myotonic dystrophy type I (DM1). We are studying repeat instability in new DM1 knockin mouse models, that contain expanded CTG repeats in the 3’ UTR of Dmpk, which show modest germline instability and parent-of-origin effects. The founder and initial progeny showed a TTG repeat interruption towards the 3’ end of the expanded CTG repeat. We observed an intergenerational expansion from 350 to 440 CTG repeats during propagation of that line, associated with loss of this interruption. Currently, these mice are being analyzed by long-read DNA sequencing for their repeat size in different tissues and age-dependent somatic instability of the repeat expansion. While recent studies support the feasibility of using long-read DNA sequencing for DM1 genetic diagnostics, our sequencing data and other reports^[1] suggest amplification bias and technical limitations of the sequencing platforms need to be addressed for making it a reliable, high-throughput analysis method. We have generated compound heterozygous mice carrying 440 CTG repeats on one Dmpk allele and 600 CTG repeats on the opposite allele, with different short sequence tags inserted adjacent to the repeats. This system allows us to directly observe and quantify size bias and PCR recombination events and optimize our amplification protocol, minimizing those measurement errors. I will be studying the possibility that amplification-based long-read DNA sequencing may provide a suitable platform for comprehensive, cost-effective genetic analysis of DM1. [1] Tsai, Y.-C., de Pontual, L., Heiner, C., Stojkovic, T., Furling, D., Bassez, G., et al. (2022). Identification of a CCG-enriched expanded allele in DM1 patients using Amplification-free long-read sequencing. bioRxiv preprint. http://doi.org/10.1101/2022.02.22.481438

16. Initial psychometric properties of the congenital myotonic dystrophy type 1 rating scale (CDM1-RS)

    N Nikolenko¹, Joe P Horrigan², MF Snape², E Fantelli², A Veerapandiyan³, S Evans², and H Lochmuller^{4
    1} National Hospital for Neurology and Neurosurgery, University College London, United Kingdom; ² AMO Pharma Ltd., United Kingdom; 3 Arkansas Children’s Hospital, Little Rock, Arkansas, USA; 4 Children’s Hospital of Eastern Ontario, Ottawa, Canada.

    Objective: The CDM1-RS is a novel rating scale for Congenital Myotonic Dystrophy Type 1 (CDM1), a rare, genetic form of muscular dystrophy. It has 11 items, each rated on a severity scale from 0 to 4. The psychometric properties of this scale have yet to be fully defined. Methodology: The clinician-completed CDM1-RS is the primary outcome measure in an ongoing phase 2/3 clinical trial in CDM1 (NCT03692312). The study is enrolling youth aged 6 to 16 y.o. in the U.S., Canada, Australia and New Zealand. It compares AMO-02/tideglusib versus placebo in a randomized, double-blinded 5-month treatment period. Clinic visits and telehealth (video) evaluations are conducted regularly. Results: At baseline, the most commonly endorsed items (n = 34) are communication difficulties (mean baseline score = 2.4, on the 0 to 4 Likert scale), difficulty thinking (2.3), and problems with hands or arms (2.07). The least endorsed items are signs of pain (0.31), breathing difficulties (0.82), and choking or swallowing issues (0.93). In-clinic scores correlate closely with telehealth-administered scores (Pearson R = 0.93, p < 0.0001), and with Clinical Global Impression–Severity scores. Clinician CDM1-RS scores correlate with caregiver-completed rating scale scores, and CDM1-RS subscale scores for cognition and ambulation correlate with functional tests (e.g. 10-meter Walk/Run test (p < 0.001)). Conclusions: The CDM1-RS is a relatively low-burden rating scale with initial evidence of sound psychometric properties. It can be administered in the clinic or remotely. It correlates well with other outcome measures, including caregiver-completed assessments, as well as with functional/ performance-based assessments.

17. AAV9-mediated overexpression of MBNL rescues DM1 cardiac phenotypes

    Rong-Chi Hu*¹ and Thomas A. Cooper^{1, 2
    1}Department of Integrative Physiology, ²Department of Pathology and Immunology, Baylor College of Medicine, Houston, Texas, USA.

    Over half of individuals affected by DM1 have cardiac involvement, such as conduction defects and arrhythmias, which can lead to sudden cardiac death, the second leading cause of death in DM1. RNA containing expanded CUG repeats (CUG_exp) transcribed from the mutant allele causes DM1 pathogenesis by disrupting functions of RNA binding protein families, such as MBNL and CELF1. While many molecular effects of CUG_exp RNA have been identified in skeletal muscle, the details of how CUG_exp RNA induces cardiac involvement are unknown. We used our DM1 heart mouse model, which expresses cardiomyocyte-specific and tetracycline-inducible RNA containing 960 interrupted CUG repeats (CUG₉₆₀) to determine the degree to which loss of MBNL activities contribute to cardiac pathogenesis by testing for phenotypic rescue. Systemic AAV9 was used for heart-specific overexpression of epitope-tagged MBNL1 and MBNL2 that was confirmed in left ventricles and atria. AAV9-MBNLs, but not the AAV9-mCherry control, significantly reduced the disrupted alternative splicing events, as well as the prolonged QRS and QTc conduction intervals induced by CUG₉₆₀ RNA. The AAV9-MBNLs cohort showed trends of rescued heart weight, ventricular wall thickness, and ejection fraction. To understand the individual contribution of MBNL1 and MBNL2, we systemically delivered single heart-specific MBNL1 and MBNL2. We found that they both significantly rescued the prolongation of QRS and QTc intervals and the heart weight, with MBNL1 showing a greater effect in QTc intervals. Overall, the data indicates that MBNL1 and MBNL2 both play a crucial role, while MBNL1 may have more influence, in DM1 cardiac pathogenesis.

18. Evaluation of in vitro gene editing removal of repeat expansion as potential treatment for myotonic dystrophy type 1

    Georgia Giannoukos*¹, Jen DaSilva*¹, Eugenio Marco Rubio*¹, Jason Arroyo*², Claudia Huichalaf*³, Sumanta Dey², Dawn Ciulla¹, James Bochicchio¹, Gregory Gotta¹, Deric Zhang¹, Tongyao Wang¹, Nicolas Christoforou³, Christopher Wilson¹, and Alain Martelli^{3
    1}Editas Medicine, Cambridge, Massachusetts, USA; ²Functional Genomics, Pfizer Inc, Massachusetts, USA; ³Rare Disease Research Unit, Pfizer Inc., Massachusetts, USA. *Equal contribution

    Myotonic Dystrophy type 1 (DM1) a multisystem rare autosomal dominant disorder; is caused by a CTG triplet repeat expansion in the 3’ UTR of the DMPK gene that leads to RNA toxic gain of function. CUG-containing transcript forms nuclear foci that sequester essential splicing factors of the Muscleblind-like family (MBNL 1, 2 and 3), affecting the splicing of multiple genes triggering cellular dysfunction and symptoms. Using CRISPR/CAS9, we aimed to remove the CUG repeat expansion present in DM1 cells as a potential therapeutic benefit. We designed 300 guides upstream/downstream of the repeat and tested them in primary human T cells to identify high editing efficiency guides. Nine pairs of the most efficient guides flanking the repeat were further assessed by electroporation into control and DM1 myoblasts, using UDiTaS sequencing to detect small indels, large deletions, inversions, large relocations, and translocations. We observed 20-30% excision of the repeat for the six most efficient pairs. To improve quantification of large deletion events, we used Oxford Nanopore long-read sequencing to measure editing with the two top pairs. Long-read sequencing revealed that 15-20-fold more editing occurred on the short compared to expanded pathogenic allele in DM1 myoblasts. To assess functional impact of repeat excision, we used Fluorescence In-Situ Hybridization (FISH) to measure RNA puncta in control and DM1 myotubes. FISH analysis immediately post-electroporation gave a strong reduction of foci, but the effect was lost upon cell expansion. It is unclear why foci reduction is transient and is presumably due to an assay artifact or by temporary reduction of transcripts by an unknown mechanism. When performing assays monitoring editing, it is important to confirm the inheritance of the phenotype.

19. Microscopy-based identification of RNA foci modulators in myotonic dystrophy type 1

    Sara Johnson and Thomas A. Cooper M.D.
    Baylor College of Medicine, Houston, Texas, USA.

    Myotonic Dystrophy Type 1 (DM1) is a multi-systemic disorder affecting 1:8000 individuals. DM1 is caused by a triplet repeat expansion within the 3’ untranslated region of the Dystrophia Myotonica Protein Kinase (DMPK) gene ranging from 5-38 repeats in unaffected individuals to as many as 5000 repeats in affected individuals. The RNA from the expanded allele (CUGexp RNA) forms a dynamic multi-branched hairpin structure that aggregates into compact nuclear foci and sequesters the Muscleblind-like (MBNL) family of splicing regulators eliciting an alternative splicing transition from adult to fetal protein isoforms in adult tissues. Despite CUGexp RNA foci being the origin of DM1 pathology, the factors that control CUGexp RNA levels and homeostasis remain largely unknown. I performed a medium-throughput small molecule screen using RNA Fluorescent In Situ Hybridization (FISH) detection of CUGexp RNA in immortalized human DM1 myoblasts to identify compounds that reduce or enhance CUGexp RNA foci. I identified and validated four compounds that significantly and robustly reduce RNA foci in two distinct DM1 cell lines, including three CDK inhibitors and one RNA polymerase II inhibitor. The foci-reducing effect of each compound is mediated through transcriptional downregulation of DMPK RNA leading to rescue of MBNL1 mis-localization and downstream DM1 spicing mis-regulation. Three additional compounds were identified that enhance foci signal intensity which inhibit two separate components involved in protein degradation. The foci-enhancing compounds function through a differentiation-dependent mechanism modulating DMPK RNA levels. Both sets of compounds provide the foundation to better understand mechanisms regulating CUGexp RNA metabolism.

20. Choroid plexus spliceopathy in myotonic dystrophy type 1

    Benjamin Kidd*^1,2, C. Nutter¹, H. Carter¹, P. Mackie², J. Sampson⁵, Ł. Sznajder¹, L. Ranum¹, E. Wang¹, H. Khoshbouei², S. Prokop^3,4, J. Day⁵, and M. Swanson^{1
    1}Department of Molecular Genetics and Microbiology, Center for NeuroGenetics, University of Florida, College of Medicine, Gainesville, Florida, USA; ²Department of Neuroscience, McKnight Brain Institute, University of Florida, College of Medicine, Gainesville, Florida, USA; ³Department of Pathology, University of Florida, College of Medicine, Gainesville, Florida, USA; ⁴Center for Translational Research in Neurodegenerative Disease, University of Florida, College of Medicine, Gainesville, Florida, USA; ⁵Department of Neurology, Stanford School of Medicine, Stanford, California, USA.

    Dmpk CTG^exp knockin (KI) mouse models for myotonic dystrophy type 1 (DM1) demonstrated the choroid plexus (ChP) is particularly susceptible to DM1 spliceopathy. The ChP is important for neurodevelopment, brain homeostasis, circadian rhythms, and sleep via its production and regulation of cerebrospinal fluid (CSF). To clarify how the ChP is affected in DM1, we investigated transcriptomic changes by RNAseq. As a first step, DM1 is characterized by impairments in developmental pre-mRNA splicing transitions regulated by the MBNL family of RNA binding proteins (RBP). Thus, we performed RNAseq on wild-type mouse ChP from late embryogenesis to adults and correlated ChP developmental splicing transitions with the corresponding changes in regulatory RBP expression. Next, we determined that both mouse Dmpk CTG^exp KI and human DM1 ChP shows a spliceopathy that primarily reverts splicing patterns to an earlier developmental pattern, and Mbnl2 KO and Dmpk CTG⁴⁸⁰ KI mice, as well as human DM1, choroid plexi show mis-splicing of important ion channels and secreted proteins. Based on these and additional findings, we propose that ChP mis-splicing leads to an alteration in CSF composition resulting in pathological features of the DM1 CNS.

21. A novel EEV-conjugated PMO, ENTR-701, reduces nuclear foci and corrects aberrant splicing in myotonic dystrophy type 1 preclinical models

    Nerissa C. Kreher, Nelsa L. Estrella, Amy N. Hicks, Xiulong Shen, Mark Wysk, Mahboubeh Kheirabadi, Matthew Streeter, Wenlong Lian, Nanjun Liu, Sara L. Blake, Christopher M. Brennan, Ning Li, Vlad Batagui, Keyede Oye, Ningguo Gao, Daniel Wang, Ziqing Leo Qian, Mahasweta Girgenrath, and Natarajan Sethuraman
    Entrada Therapeutics Inc, Boston, Massachusetts, USA.

    Objective: Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy and is caused by a CUG trinucleotide repeat expansion in the DMPK (dystrophia myotonica protein kinase) mRNA. This mutant mRNA sequesters RNA binding proteins such as MBNL (Muscleblind Like Splicing Regulator) proteins in the nucleus, leading to dysregulation of alternative splicing across several organs. There are currently no approved therapies for DM1. One potential therapeutic approach for DM1 is selective blockade of CUG repeats with an oligonucleotide. Methodology: We developed a family of proprietary cyclic cell-penetrating peptides that form the core of our Endosomal Escape Vehicle (EEVTM) technology, which facilitates intracellular delivery, endosomal escape, and localization of therapeutics to the nucleus. ENTR-701 is our lead clinical candidate for DM1 and is composed of our EEV technology, optimized for skeletal and cardiac muscle functional delivery, conjugated to a PMO (phosphorodiamidate morpholino oligomer) that specifically binds and sterically blocks interactions between the CUG repeats and RNA binding proteins in an allele-specific manner. Results: ENTR-701 significantly reduced nuclear foci and corrected splicing defects in DM1 patient-derived muscle cells, genetically modified HeLa cells, and the HSA-LR mouse model of DM1. In addition, ENTR-701 selectively reduced mutant transcript levels in both HeLa cells and HSA-LR mice. Furthermore, a single dose of ENTR-701 corrected splicing defects and myotonic phenotype in HSA-LR mice for up to 8 weeks post dose. Conclusions: These results illustrate the therapeutic potential of the EEV-oligonucleotide approach for DM1, and support further study of ENTR-701 in patients with DM1.

22. Extracellular RNA splice events in cerebrospinal fluid as candidate biomarkers of myotonic dystrophy

    Preeti Kumari*, Ningyan Hu, Alex Sizemore, Lauren Sullivan, Brigham McKee, Parker Conquest, Thurman Wheeler
    Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA.

    Objective: To evaluate extracellular RNA (exRNA) in cerebrospinal fluid (CSF) as a source of CNS-derived splice events that may serve as biomarker of DM1 disease activity. Methodology: We examined fresh CSF samples (N = 14) from the MGH CSF biobank. To separate EVs from cells contained in CSF, we used low speed centrifugation followed by filtration of the supernatant. EV size and concentration were measured using microscopy and particle tracking analysis software (NanoSight). We extracted ExRNA from the EV pellet, produced cDNA, and quantified gene expression and splice events (% exon inclusion) by droplet digital PCR. Results: Mean particle diameter is about 175 nm in CSF vs. 220 nm in urine. In CSF cells, total RNA content is 10 - 100-fold higher and splicing patterns for transcripts MBNL2, NCOR2, MAP3K4, and GOLGA4 significantly different as compared to CSF exRNA. Normalized expression of both DMPK and CNBP was 50 - 60% higher in CSF exRNA than in CSF cells. Conclusions: Quantification of CNS-derived alternative splice products in CSF exRNA is feasible and we expect will show a differential pattern in DM as compared to non-DM. Removal of leukocytes and erythrocytes normally found in CSF will enhance accurate quantification of CNS-derived splice events. Funding: Elaine and Richard Slye Fund; Myotonic Dystrophy Foundation.

23. Identifying novel metabolic targets for myotonic dystrophy type 1

    Dhvani H Kuntawala¹, Rui Vitorino¹, and Sandra Rebelo^{1
    1}Medical Sciences Department, Institute of Biomedicine – iBiMED, University of Aveiro, Aveiro, Portugal.

    Myotonic dystrophy type 1 (DM1) is an autosomal dominant hereditary disease caused by abnormal expansion of unstable CTG repeats in the 3’ untranslated region of Myotonic Dystrophy protein kinase (DMPK) gene being the most common muscular dystrophy among adults⁽¹⁾. Clinical features observed in patients with DM1 include myotonia, muscle weakness, and atrophy. Additional clinical features observed are cardiac and respiratory complications, sleep disorder, dyslipidemia, insulin resistance and also gastrointestinal and central nervous system alterations ^(2-3). Despite, some studies reporting therapeutic strategies for the treatment of DM1, many issues remain unsolved such as the contribution of metabolic and mitochondria dysfunctions for DM1 pathogenesis. Thus, it is imperative to identify novel metabolic targets for DM1. Resorting bibliometric analysis, articles combining DM1, and metabolic/metabolism terms were identified and further analyzed using the Vosviewer software. A list of potential molecular targets for DM1 associated to metabolic/metabolism was generated. This gene list was compared with genes previously associated with DM1 in DisGeNET database. The results revealed that only 15 were common between Vosviewer and DisGeNET analysis. Of note, we identified 60 genes not previously associated with DM1 in DisGeNET, but they are of interest and will be further evaluated for unravelling the contribution of metabolic alterations for DM1 pathogenesis. Through molecular techniques, validation of some potential metabolic targets will be achieved. In overall, our study has strengthened the hypothesis that metabolic and mitochondria dysfunctions might contribute for DM1 pathogenesis and the exploitation of the metabolic dysfunctions as target for development of therapeutic interventions for DM1. References: 1. Darras Basil T. Myotonic dystrophy: Etiology, clinical features, and diagnosis. UpToDate. 2009.; 2. Meola G, Cardani R. Myotonic dystrophies: An update on clinical aspects, genetic, pathology, and molecular pathomechanisms. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease. 2015;1852(4):594-606.; 3. Johnson N, Aldana E, Angeard N, Ashizawa T, Berggren K, Marini-Bettolo C et al. Consensus-based care recommendations for congenital and childhood-onset myotonic dystrophy type 1. Neurology: Clinical Practice. 2019;9(5):443-454. Acknowledgments: This research was funded by Fundação para a Ciência e a Tecnologia (FCT) through the Institute of Biomedicine (iBiMED) UIDB/BIM/04501/2020/UIDP/04501/2020 and by the MEDISIS project (CENTRO-01-0246-FEDER-000018).

24. A CTG repeat-selective screen of a natural product library reveals dietary natural compounds as potential therapeutics for myotonic dystrophy.

    Subodh Kumar Mishra*¹, Sawyer Hicks¹, Tammy Reid ¹, John D. Cleary¹, Kaalak Reddy¹, Masayuki Nakamori², and J. Andrew Berglund^{1
    1}The RNA Institute, College of Arts & Sciences, University at Albany, State University of New York, Albany, New York, USA; ²Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan.

    Objective: Myotonic dystrophy (DM) is a multisystemic neuromuscular disease caused by the expansion of CTG or CCTG repeat tracts in non-coding regions of DMPK and CNBP genes respectively. Blocking transcription of these repeat expansions is a promising therapeutic approach for mitigating toxic RNA-associated pathogenesis[1]. The current study identified a new class of dietary natural compounds that exhibit promising therapeutic potential for the treatment of DM. Methodology: We utilized our previously developed DM1 HeLa cell model to screen a natural product library obtained from NCI [1]. The activity of lead compounds from this screen was then validated in DM patient-derived fibroblasts, myotubes, and the DM1 HSA^LR mouse model. Results: Primary screening revealed NP1-NSA1 as a selective modulator of toxic CUG RNA abundance, as it reduced r(CUG)₄₈₀ level by ~50%. This compound is abundantly present in several fruits and vegetables that are part of the daily human diet. NP1-NSA1 notably rescued DM-associated mis-splicing events in both DM1 and DM2 patient-derived fibroblast cell lines and in DM1 patient-derived myotubes. NP1-NSA1 also reduced HSA CUG transgene expression and rescued mis-splicing in the DM1 HSA^LR mouse model without major transcriptomic dysregulation. NP1-NSA1 also improved myotonia in DM1 HSA^LR mice without any observable toxicity. Conclusions: The excellent safety profile of NP1-NSA1 and its efficacy in multiple DM model systems with little to no adverse effects position this natural product as a lead compound for therapeutic consideration in DM. References: Reddy, K., et al., A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels. Proceedings of the National Academy of Sciences, 2019. 116(42): p. 20991-21000.

25. Specialists involved in the diagnosis and management of myotonic dystrophy based on a real-world data analysis pre-and post-diagnosis

    John W. Day¹, Kathryn A. Munoz², Richard A. Brook³, Nathan L. Kleinman³, Chao-Yin Chen², Brad McEvoy², Kelly DiTrapani², Nathan L. Kleinman³, Mark Stahl², Li-Jung Tai²1Stanford Neuroscience Health Center, Stanford, California, USA; ²Avidity Biosciences, Inc., San Diego, California, USA;³Better Health Worldwide, Newfoundland, New Jersey, USA.

    Objective: Describe the types of specialists involved in identifying and managing patients with myotonic dystrophy (DM) compared with matched controls (MCs) two years before and after diagnosis. Design/Methods: We used PharMetrics deidentified-US-claims (Jan-2010—Mar-2021) to retrospectively evaluate care for DM-patients vs non-DM MCs. DM-patients were required to have ≥2 DM claims ≥30 days apart (index date=first diagnosis date). Five non-DM controls were matched to each DM-patient. Costs, days of care, and types of specialists were compared two-years post-diagnosis minus two-years pre-diagnosis (Post-PreDx). Data reported are per-member-per-year for costs, number of services, and days of service. Costs were adjusted to 2020 dollars. All reported findings significant (p < 0.05). Results: We identified 519 DM-patients and 2,595 MCs. Most outcomes demonstrated higher care utilization in DM-patients than MCs. Post-PreDx medical care days increased at all service settings for both cohorts (↑9.9DM-vs-↑1.5MCs) except lab. Total medical and drug costs increased more for DM-patients ($18,705 to $25,594) than MCs ($5,640 to $6,684). Pre-diagnosis, DM-patients collectively received care from multiple specialties including neurology, cardiology, ophthalmology/optometry, radiology, gastroenterology, primary care, and emergency medicine, more frequently than MCs. Post-diagnosis care from neurologists, cardiologists, pulmonologists, and internal medicine increased significantly more in DM-patients than in MCs. Conclusions: Healthcare costs/care days increased significantly in DM-patients following diagnosis and were received from different specialists than MCs. This likely reflects clinicians diagnosing and managing DM and related conditions leading to and following a formal diagnosis. Since there are no specific treatments for DM, there is a high unmet need for disease-modifying therapies.

26. Alternative splicing mediates the compensatory upregulation of MBNL2 upon MBNL1 loss-of-function

    Larissa Nitschke*¹, Rong-Chi Hu², Andrew N. Miller¹, and Thomas A. Cooper^{1,2,3
    1}Department of Pathology & Immunology, Baylor College of Medicine, Houston, Texas, USA; ²Department of Molecular Physiology & Biophysics, Baylor College of Medicine, Houston, Texas, USA; ³Department of Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

    Objective: The effect of pathogenic genetic variations can be compensated by paralogs with redundant functions. An example of such compensation is found with the paralogs of the Muscleblind Like (MBNL) family of RNA binding proteins. Loss of MBNL1 increases MBNL2 in tissues where Mbnl2 expression is low. As such, Mbnl1^-/- mice develop relatively mild phenotypes, while Mbnl1^-/-; Mbnl2^+/- mice display severe phenotypes recapitulating Myotonic Dystrophy Type 1 (DM1), a multisystemic disorder in which an expanded CUG RNA repeat sequesters the MBNL paralogs. The mechanism by which MBNL2 is upregulated and the impact on DM1 pathogenesis remain unknown. Methodology: In this study, we used molecular and cellular assays upon Mbnl1 knockdown and knockout in cell culture and in vivo to uncover the mechanism by which loss of Mbnl1 upregulates MBNL2 levels. Results: We found that loss of Mbnl1 upregulates MBNL2 via the enhanced inclusion of Mbnl2 exon 9, that introduces an alternative C-terminus. We show that the C-terminus excluding exon 9 drastically destabilizes MBNL2. Thus, the inclusion of exon 9 upregulates MBNL2 via a switch in C-termini and the loss of protein degradation signal. Lastly, we found that inclusion of Mbnl2 exon 9 is increased in a DM1 mouse model in which MBNL2 protein levels are upregulated. Conclusions: This study uncovered the mechanism by which loss of MBNL1 upregulates its paralog MBNL2 and suggests that the compensatory mechanism is active in DM1. Future work will investigate the importance of the compensatory mechanism in DM1 and explore its utilization for therapeutic purposes.

27. TREAT-NMD Myotonic Dystrophy Global Registry Network: An international collaboration in myotonic dystrophy type 2

    Peric S¹, Porter B¹, Bennett N¹, Allison D¹, Ashley E-J¹, Esparis B¹, Campbell C¹, Guglieri M¹, Ambrosini A¹, Roxburgh R^{1
    1}On behalf of TREAT-NMD Myotonic Dystrophy Subgroup & TREAT-NMD Global Registry Network

    Introduction: Myotonic dystrophy type 2 (DM2) is a rare multi-system disease recognised in the last three decades. The TREAT-NMD Global Registry Network is a global collaboration of registries collecting data on neuromuscular conditions such as DM2. The aim is to assess the number of DM2 patients included in the network, and analyse socio-demographic/clinical features. Methods: An email survey sent to the 22 member DM registries requested data on number of DM1 and DM2 patients, population catchment, and clinical features. Results: Of 13 DM registries that responded, eight enrolled DM2 patients. Total number of DM2 cases was 1,720, with the Czech/Slovakian, German and the USA (MDF) registries enrolling most patients, with 445, 430, and 339, respectively. The highest number of registered cases per 100,000 population was seen in the Czech/Slovakia (4.2) and Serbia (2.0). The DM2:DM1 ratio was highest in the Central European countries. Registry enrolment occurred at a median age of 51 years with 63% being female. Onset of DM2 occurred before the age of 20 in 14% of cases. One fifth of DM2 patients used an assistive device to walk and 4% were non-ambulant. Pacemaker or implantable cardioverter-defibrillator was reported in 4% of DM2 subjects, while 7% used non-invasive ventilation. Conclusions: TREAT-NMD member registries were able to assemble the largest DM2 cohort to date with an international reach, providing meaningful clinical and demographic data. More DM registries should aim to capture DM2 data to contribute to important collaborations like this, which can support future research and clinical trial recruitment.

28. Injury among adults with myotonic dystrophy type 1: Findings from linkage of South Carolina and Utah’s muscular dystrophy surveillance, tracking, and research network data to statewide emergency department encounters

    Reeder Matt R¹, Royer J², Butterfield RJ³, McDermott S⁴.
    ¹Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, Utah, USA; ²South Carolina Revenue and Fiscal Affairs, Columbia, South Carolina, USA; ³Department of Pediatrics and Neurology, University of Utah, Salt Lake City, Utah, USA; ⁴City University of New York, School of Public Health and Health Policy, New York, New York, USA.

    Objective: Adults with myotonic dystrophy type 1 (DM1) may be at increased risk for injury due to their muscle disease symptoms, but the injury experience of this population is not well understood. Causes and types of injury identified by emergency department (ED) encounters among adults with DM1 residing in South Carolina (SC) and Utah (UT) were examined. Methodology: Adults (>18 years) with DM1 from SC and UT were identified by MD-STARnet surveillance and linked to each site’s statewide ED data from 1/1/2008-12/31/2016. Causes and types of injuries were determined from applying ICD injury matrices to diagnosis codes. Aggregate data from the two states were combined and yearly rates for all-cause and injury-related ED visits were averaged. The distribution of causes and types of injury were assessed. Results: We linked 83 adults with DM1 (nearly 50% 18-39 years old, 55% female, 89% white, 76% non-Hispanic/unknown ethnicity). Between 2008-2016, 220 ED visits (48 injury-related) were identified. Average yearly visit rates were 295 per 1000 population (all-cause ED) and 64 per 1000 population (injury-related). Reasons for injury-related ED visits included falls (44%), motor vehicle traffic related (17%), and other causes (33%). Injuries were comprised of sprains/strains (27%), contusions (21%), and other types (52%). Conclusions: Understanding injury experiences of people with DM1 may help develop prevention strategies for at-risk patients. Future studies comparing these results to age-specific general-population rates can identify if individuals with DM are at higher risk for injury.

29. TREAT-NMD Myotonic Dystrophy (DM) Global Registry Network: An update in 2022

    Roxburgh R¹, Porter B¹, Bennett N¹, Walker H¹, Allison D¹, Ashley E-J¹, Esparis B¹, Campbell C¹, Guglieri M¹, Ambrosini A¹, Peric S^{1
    1}On behalf of TREAT-NMD Myotonic Dystrophy Subgroup & TREAT-NMD Global Registry Network.

    Introduction: TREAT-NMD is an international collaboration that aims to accelerate the development of new treatments for neuromuscular diseases. The TREAT-NMD DM Global Registry Network/Subgroup was established in 2017 specifically to develop collaborations amongst member DM registries. Registries collect an agreed minimum dataset, with some collecting additional data. We present an update of clinical data from the network. Methods: An email survey was sent to the 22 member DM registries, requesting data on demographics, and respiratory and cardiac measures in DM1 patients. Results: Responses were received from 13 / 22 registries. Registry enrolments ranged from 13 patients to 1,459, with a total of 6,472 patients. Among nationally recruiting registries, average cases per 100,000 was 2.53. Non-ambulant patients represented 8.8% of registry patients on average and a further 19.5% required a walking aid. Cardiac conduction defects were reported in 33.8% of patients on average but only 7.9% (range of 3.1% - 16.0%) were fitted with a pacemaker or implantable defibrillator. Daytime sleepiness was present on average in 73.2% of patients, with 13.8% having FVC < 50% expected, 13.8% using non-invasive ventilation and 1.3% using invasive ventilation. On average 3.4% of patients used a feeding tube. Patients’ data are updated annually for 9 of the 13 registries, and as required for enquiries for the remainder. Conclusions: This survey reveals the considerable burden of disease related to DM. It also reveals some inter-country differences which is especially useful for discussing best practice. Registry data should be updated regularly, ensuring that it is accurate for such analyses.

30. Circadian rhythm disruptions in myotonic dystrophy type 1 (DM1) – a multi-systemic genetic disease with major CNS involvement

    Zoe J. Scherzer*¹, Belinda S. Pinto¹, Miguel Gutierrez², Karyn Esser², and Eric T. Wang¹.
    ¹Molecular Genetics and Microbiology Department, University of Florida, USA; ²Department of Physiology and Functional Genomics, University of Florida, USA.

    Myotonic Dystrophy Type 1 (DM1) is caused by the expansion of CTG repeats in the 3’ UTR of the DMPK gene. Expression of these expanded repeats produces a toxic RNA that sequesters the Muscleblind-like (MBNL) splicing factor into nuclear foci resulting in global splicing misregulation. While DM1 is known as a muscular dystrophy, patients show a range of CNS symptoms including hypersomnolence and sleep dysregulation. A core clock gene, Casein Kinase 1 Delta (CSNK1D), is misspliced in multiple tissues of DM1 patients and mouse models, with potential impacts on sleep/wake rhythms. Thus, we are investigating whether circadian disruption contributes to DM1 pathology, by examining the effect of the DM1 mutation on circadian rhythms in cell and mouse models of the disease. We have shown that MBNL or presence of repeats have a direct and opposite effect on splicing of a CSNK1D alternative exon in cell culture. Significantly, this alternative splicing event dictates formation of two different period-controlling CSNK1D isoforms. We have also performed circadian activity analyses of DM1 mouse models that either express expanded CTG repeats or lack MBNL function and display muscle or CNS phenotypes characteristic of the disease. Interestingly, all of these models, while maintaining normal levels of overall activity, display activity rhythms of reduced amplitude. In addition, the DMPK 480 CTG knockin model, which primarily displays nuclear RNA foci in the CNS with a prominent presence in the choroid plexus (CP), shows a mild shortening of the circadian period. These data suggest that disruption of central and peripheral clocks contribute to altered circadian rhythms in DM1. We will further extend these observations by examining circadian rhythms in CNS and muscle tissues using the PER2:LUCIFERASE reporter and transcriptomic analyses. Taken together, our data show that circadian rhythms are disrupted in DM1 models and future studies will provide key insights into how circadian disruption contributes to DM1 pathology.

31. Single-administration of a cyclic peptide-conjugated CUG-repeat steric blocker rescues myotonia and molecular phenotypes in HSALR mice

    Emma N. Shea^1,2, M. Carmen Valero², Marina M. Scotti², Derek R. Muscato², Leanne M. Adams², Xiulong Shen³, Mahboubeh Kheirabadi³, Mark Wysk³, Matthew Streeter³, Natarajan Sethuraman³, Ziqing Qian³, and Eric T. Wang^{2
    1}Biomedical Sciences Graduate Program, University of Florida, Gainesville, Florida, USA; ²Department of Molecular Genetics and Microbiology, Center for NeuroGenetics, Genetics Institute, University of Florida, Gainesville, Florida, USA; ³Entrada Therapeutics, Boston, Massachusetts, USA.

    In Myotonic Dystrophy type 1 (DM1), expanded CUG repeats in the DMPK transcript sequester MBNL splicing proteins. This causes molecular phenotypes including nuclear foci accumulation and aberrant splicing, leading to symptoms such as myotonia and muscle weakness. Multiple oligonucleotide therapeutics have been developed to either cleave DMPK mRNA or displace MBNL from the CUG repeats, but a major barrier to successful implementation of these approaches is efficient delivery to muscle tissue. Conjugation of oligonucleotides to antibodies, antibody fragments, and peptides to enhance delivery efficiency are currently in development by several groups. Here, we show that cyclic peptide oligonucleotide conjugates efficiently displace MBNL from expanded CUG repeats in human DM1 cells and mouse models of DM1. Nuclear CUG repeat foci were reduced in a CUG-repeat knock-in cell line (HeLa480) and patient-derived myoblasts, and splicing events were also assayed and rescued in HeLa480. A single intravenous administration into HSA^LR mice eliminated myotonia one week after injection. RNA FISH in tibialis anterior myofibers showed a reduction in nuclear CUG RNA foci. Aberrant splicing patterns of Mbnl1, Atp2a1, and Nfix were nearly completely rescued in gastrocnemius, quadriceps, and tibialis anterior, as assayed by RT-PCR, and RNAseq analyses of gastrocnemius also confirmed global splicing correction. This study demonstrates the promise and potential of cyclic peptide oligonucleotide conjugates for the treatment of DM1.

32. Elimination of myotonia from mouse models of myotonic dystrophy type 1

    Matthew T. Sipple¹, Lily A. Cisco¹, Katie M. Edwards¹, Matthew K. Tanner², Charles A. Thornton² and John D. Lueck^{1,2
    1}Department of Pharmacology and Physiology and ²Department of Neurology, University of Rochester Medical Center, Rochester, New York, USA.

    Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy with muscular symptoms, including myotonia, weakness, and wasting, as well as many extra-muscular implications. DM1 patients possess >50 (CTG) repeats in the 3’ UTR of the DMPK gene and transcribe toxic RNA that sequester RNA-binding proteins, such as the Muscleblind-like (MBNL) family of splicing factors. This leads to widespread changes in alternative splicing. For example, increased exon 7a inclusion in transcripts of ClC-1, the main voltage-sensitive chloride channel on skeletal myofibers has been linked to myotonia. In patients, the distribution of myotonia correlates with weakness and wasting in various muscle groups. Additionally, myotonia alone has been shown to cause changes in gene expression. Therefore, we hypothesize that myotonia plays a central role in driving the other skeletal muscle pathology in DM1. Furthermore, no standard disease modifying therapy exists to improve DM1 patient outcomes. We propose long-term anti-myotonic therapy could have myo-protective benefits. To investigate this, we developed a novel mouse line resistant to myotonia caused by DM1 to eliminate myotonia from models of DM1. We crossed this mouse line with the Mbnl1^-/- model of DM1 to eliminate myotonia and compare the resultant changes in muscle physiology, histopathology, and genetic regulation between myotonic and non-myotonic progeny. We have eliminated myotonia in Mbnl1^-/- mice and have observed significant physiologic, histologic, and splicing differences with the non-myotonic mice featuring phenotypes closer to wild-type. To translate this to a clinically relevant intervention, we will use pharmacologic suppression of myotonia in DM1 mice.

33. Analysis of DMPK expansion transcript degradation in DM1

    Xiaomeng Xing*¹, Amy Lennon¹, and J David Brook^{1
    1}Institute of Genetics, School of Life Sciences, University of Nottingham, UK.

    Objective: The pathogenesis of DM1 is associated with nuclear retained mutant expansion DMPK mRNA. However, it is not clear what happens to the expansion DMPK mRNAs after the dissolution of nuclear foci, and which enzymes are responsible for the degradation of them. One of our goals is to address this knowledge gap. Methodology: We seek to determine the key nucleases responsible for the degradation of expansion DMPK RNAs using shRNA expression in DM1 cell lines and their derivatives. Stochastic optical reconstruction microscopy (STORM) has been applied to discover micro foci or break-down products of the DMPK mRNAs. Results: We have successfully generated MBNL-deficient DM1 cell lines and shown loss of nuclear foci and dramatically reduced levels of expDMPK transcript retention in the resulting cells. Moreover, the STORM experiments indicate that MBNL proteins strengthen the retention of mutant DMPK transcripts and with the absence of MBNL, mutant DMPK transcripts dissipate rapidly with micro foci as intermediate degradation products. Thus, MBNL proteins play a role in inhibiting the degradation of mutant expDMPK transcripts. UPF1 inhibition produced a slight but significant increase in the number of foci in DM1 and an even greater rise in MBNL deficient DM1 cells. It indicates UPF1 plays a key role in the degradation of expDMPK mRNAs and the absence of MBNL has a profound effect on this. Conclusions: Our analysis suggests that the absence of MBNLs alleviate the retention of expDMPK transcripts in the nucleus and modulates the break-down of expanded CUG repeats in the DMPK mRNA. Moreover, UPF1 plays a key role in the degradation expDMPK transcripts and MBNLs impede the degradation of expDMPK transcripts by UPF1.

34. Building connections to reduce barriers to therapies

    Allison Formal, Nadine Ann Skinner, and Tanya Stevenson
    Myotonic Dystrophy Foundation, Oakland, California, USA.

    The Myotonic Dystrophy Foundation’s (MDF) mission is to support and connect the myotonic dystrophy community in order to accelerate research towards treatments and a cure. To accomplish this, MDF provides resources and advocacy efforts to ensure there is focus on the care and a cure for those with myotonic dystrophy. The MDF builds collaborations and knowledge through a global network and alliance of DM academic and industry researchers that breaks down barriers to understanding the disease and developing treatments and a cure. In seeking to de-risk the path to a cure, MDF engages in strategic funding of early stage-science (cell lines, gene editing), the career development of pre- and post-doctoral scientists, and transformative clinical research studies. MDF additionally supports the creation and development of the Myotonic Dystrophy Research Map that enables the entire DM community stakeholders and its eco-system to understand the connections and their contributions to further de-risking the science, building global advocacy, and enhancing patient registries and outcomes. MDF tracks the results of these efforts to better understand the impacts these de-risking strategies have on accelerating treatments and a cure.

35. Myotonic Dystrophy Family Registry

    Niv Joshi, Kleed Cumming, and Tanya Stevenson
    Myotonic Dystrophy Foundation, Oakland, California, USA.

    In 2013, the Myotonic Dystrophy Foundation (MDF) launched the Myotonic Dystrophy Family Registry. The purpose of the Myotonic Dystrophy Family Registry (MDFR) is to: 1) assist researchers seeking participants for clinical trials and research studies, 2) speed up research in myotonic dystrophy (DM) by collecting information that scientists can use, 3) communicate with the DM community about upcoming research studies and clinical trials, 4) understand how DM can affect the quality of life and circumstances of people and families living with it, 5) help medical professionals improve how they treat individuals affected by DM, and 6) learn how and why certain treatments work and don’t work. The MDFR currently has 2,424 registrants from 55 countries supporting the work of DM research globally.

36. The Global Alliance for Myotonic Dystrophy Awareness efforts to launch International Myotonic Dystrophy Awareness Day

    Kate Beck, Niv Joshi, Kleed Cumming, MK, and Tanya Stevenson
    Myotonic Dystrophy Foundation, Oakland, California, USA.

    The Global Alliance for Myotonic Dystrophy Awareness, which includes over 50 international nonprofit organizations, academic and research institutions, biotechnology and pharmaceutical companies, patient advocacy groups, and others focused on raising global awareness about myotonic dystrophy, united for the first time on Rare Disease Day in February of 2021 to declare International Myotonic Dystrophy Awareness Day be observed each September 15^th. The purpose of raising DM awareness is to improve quality of life, the availability of essential resources, access to appropriate healthcare for individuals living with DM, and to help accelerate the research and drug development process by promoting understanding and support for the community across a broad range of people – general public, policy makers, regulators, biopharmaceutical representatives, researchers, health care professionals, and anyone with an interest in changing the future of myotonic dystrophy. In 2021 the Global Alliance focused awareness efforts on establishing International Myotonic Dystrophy Awareness Day be observed each September 15^th. The efforts included the adoption of the official International Myotonic Dystrophy Awareness Day logo, endorsements by federal and state governments and government officials, nearly 50 US and UK monuments, landmarks, and buildings lit in green, and grassroots awareness campaigns on social media and beyond. The Global Alliance continues its efforts in 2022 by promoting general awareness while collectively focusing efforts on raising DM awareness amongst clinical care teams and preparing participants for clinical trials.