Research Posters - 2021 Virtual 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 as well as to provide unopposed time for researchers to discuss their findings in a Zoom-like format with conference attendees, including other researchers, clinicians, individuals with DM, and their families. Click here to learn more about the 2021 Virtual MDF Annual Conference.

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

1. Towards the identification of biomarkers of muscle function improvement in myotonic dystrophy type 1.
2. Targeting defective muscle stem cells as a potential therapeutic approach for myotonic dystrophy type 1.
3. Enhanced anesthetic, benzodiazepine sensitivity, and GABAAR mRNA dysregulation in a MBNL2 knockout mouse model of myotonic dystrophy.
4. Generation and characterization of a DM2 BAC mouse model.
5. Structural brain abnormalities in adult-onset myotonic dystrophy type 1.
6. Introducing a guide in physiotherapy addressing sexual function in adults with neuromuscular disorders.
7. Investigating subcellular RNA localization in congenital DM1 patient iPSC-derived neurons and cortical organoids.
8. Mechanisms of DM1 cardiac pathogenesis.
9. Developing novel robust myotonic dystrophy brain biomarkers: A machine learning approach using diffusion tensor imaging data.
10. Choroid plexus dysfunction and cerebral atrophy in DM1.
11. A CTG repeat-selective screen of a natural product library reveals dietary natural compounds as potential therapeutics for myotonic dystrophy.
12. Blockmir induced MBNL rescue in DM1 models.
13. MBNL loss of function in visceral smooth muscle as a model of myotonic dystrophy type 1.
14. Highly specific and efficient therapeutic strategy by DMPK promoter silencing in myotonic dystrophy type 1.
15. How no-amplification Single-Molecule Real-time sequencing can improve the prognosis and genetic counseling in DM1 patients.
16. The FORCE^TM platform achieves robust knock down of toxic human nuclear DMPK RNA and foci reduction in DM1 cells and in newly developed hTfR1/DMSXL mouse model.
17. Small molecule GeneTACs reduce toxic nuclear foci and correct splicing defects in multiple myotonic dystrophy type 1 (DM1) cell types.

 

Research Poster Presentation Abstracts

Posters are listed in alphabetical order by first author. Corresponding author is presented in bold. An * denotes a current MDF Fellow.

 

1. Towards the identification of biomarkers of muscle function improvement in myotonic dystrophy type 1.

Aoussim, Amira^1,2, Marie-Pier Roussel^1,2, Anne-Marie Madore¹, Mathieu Morissette^3,4, Catherine Laprise¹, and Elise Duchesne¹. Affiliations: ¹Université du Québec à Chicoutimi, Québec, Canada, ²Lac-Saint-Jean, Hôpital de Jonquière, Québec, Canada, ³Université Laval, Québec, Canada, ⁴Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ). “Towards the identification of biomarkers of muscle function improvement in myotonic dystrophy type 1.”

Myotonic dystrophy type 1 (DM1) is the most common myopathy in adults. Skeletal muscle is severely impaired; individuals with DM1 suffer from muscle atrophy and experience a progressive loss of maximum muscle strength. We recently showed significant gains in muscle strength and functional capacity in men with DM1 who participated in a 12-week supervised strength-training program. To identify the underlying mechanisms involved in those clinical gains, a study of muscle proteome was carried out. Objectives: 1) To identify the proteomic biomarkers associated with the positive response to strength training in DM1, and 2) to develop linear statistical models explaining the modulation of muscle proteome. Methodology: An ion library was developed from the proteomic analysis of muscle biopsies collected in 11 individuals with DM1 pre- and post-training to identify the proteins that were significantly modulated by the program. The identified proteins were then classified using UniProt, Pubmed, PANTHER and REACTOME classification tools. Results: From a total of 572 proteins identified, the level of 44 was significantly modulated by the training (paired t-test p<0.05). Several functional classes in which these proteins are involved were linked to exercise-induced response, including metabolism, myogenesis and muscle contraction. Explanatory models identified morphological factors of muscle fibers as variables influencing proteome modulation. Conclusion: By investigating/targeting the biological processes and molecular functions involved in the strength-training-induced muscle gains, proteomic analysis can lead to the identification of muscle biomarkers, which could be used in future clinical trials to measure the response to a new therapy.

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2. Targeting defective muscle stem cells as a potential therapeutic approach for myotonic dystrophy type 1.

Conte, Talita¹*, I Mokhtari², Zakaria Orfi¹, Marie-Pier Roussel², Paul Fabre¹, Thomas Molina¹, Junio Dort¹, Ornella Pellerito¹, Christian Beauséjour¹, Elise Duchesne², and Nicolas Dumont¹. Affiliations: ¹CHU Sainte Justine, Montreal, Canada, ²Université de Québec à Chicoutimi, Canada. “Targeting defective muscle stem cells as a potential therapeutic approach for myotonic dystrophy type 1.”

Myotonic dystrophy type 1 (DM1) is one of the most frequent myopathies in humans, it can affect 1 person out 8,000 worldwide, but it reaches 1 out 475 people in the Saguenay–Lac-St-Jean region of Quebec, in Canada. DM1 affects particularly skeletal muscle tissue leading to progressive muscle weakness and atrophy. Thus there is an urgent need to develop novel therapeutic avenues for this disease. In DM1, muscle stem cells (MuSC) exhibit premature senescence, decreased proliferative capacity as well as delayed differentiation and fusion into myotubes. We hypothesize that the removal of these defective MuSC will restore the function of myogenic cells and skeletal muscles affected by DM1.We collected muscle biopsies from 12 DM1 patients and healthy controls to isolate myogenic cells. We performed an extensive screening with different drugs targeting the removal of defective cells and we observed that one compound showed promising results eliminating specifically defective MuSC on DM1 myogenic cell lines restoring their myogenic capacity. Since this is a translational research project, we are determining the therapeutic potential of our lead compound in a mouse model of DM1 (DMSXL mice). We are validating and comparing the mechanisms leading to defective cell function in both human and mouse cell lines and tissue. We are treating DMSXL mice with our lead compound and we will investigate the impact on skeletal muscle growth, function, and regeneration. This novel class of drugs has shown promising results in other degenerative conditions, but it has not been explored in muscular diseases.

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

Edokpolor, Kamyra^1*, Anwesha Banerjee¹, Zachary T. McEachin¹, Eric T. Wang², Paul S Garcia³, and Gary J. Bassell¹. Affiliations: ¹Emory University School of Medicine, Atlanta, Georgia, USA, ²University of Florida-Gainesville, USA, ³Columbia University, New York, New York, USA. “Enhanced anesthetic, benzodiazepine sensitivity, and GABAAR mRNA dysregulation in a MBNL2 knockout mouse model of myotonic dystrophy.”

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 GABAA extra-synaptic receptors (GABAAR.) Loss of Muscleblind-like protein 2 in mice results in CNS symptoms that phenocopy DM1, as well as various pre-mRNA mis-splicing events, such as increased production of the 2S isoform of GABRG2, which encodes the γ2 subunit of GABAA receptors. Gamma 2S-containing GABAARs are localized to extrasynaptic sites, exhibit enhanced GABA transmission and reduced desensitization, and are more sensitive to benzodiazepines (positive allosteric GABAAR modulators, PAM). Thus, we hypothesized that alterations in GABAAR subunit expression and sensitivity may underlie anesthesia-related CNS symptoms in DM1. Here, we confirm via rt-qPCR that γ2S vs. γ2L ratios are elevated in Mbnl2 KO brain.By quantitating abundance of additional GABAAR mRNAs, we also find elevation of Gabra1 mRNA in Mbnl2 KO mice, the major GABAAR subunit in brain, suggesting consequences for total levels of GABAARs. We further test the hypothesis that MBNL2 depletion leads to enhanced GABA sensitivity using behavioral paradigms. Sevoflurane, an anesthetic predominantly mediated by GABAAR, diazepam (PAM), and Zolpidem (GABAA-a1R antagonist) were administered to Mbnl2 KO mice. Delayed emergence and recovery behaviors 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 and anesthesia in myotonic dystrophy.

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4. Generation and characterization of a DM2 BAC mouse model.

Engelbrecht, Avery¹, Kiru Thangaraju¹, John Cleary¹, Tao Zu¹, and Laura P.W. Ranum¹. Affiliations: ¹University of Florida, Gainesville, USA. “Generation and characterization of a DM2 BAC mouse model.”

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are multisystemic diseases caused by CTG•CAG (DM1) or CCTG•CAGG (DM2) repeat expansion mutations located in the dystrophia myotonica protein kinase (DMPK) or cellular nucleic acid binding (CNBP) genes, respectively. While RNA gain of function effects play a role in disease, bidirectional transcription and repeat associated non-ATG (RAN) translation may also contribute to DM. RAN translation of sense (CCUG) and antisense (CAGG) expansion transcripts produces sense (LPAC) and antisense (QAGR) RAN proteins that accumulate in DM2 human autopsy brains. LPAC is found in neurons, astrocytes, and glia in gray matter while QAGR is found in oligodendrocytes located in white matter. Both LPAC and QAGR RAN proteins, which are toxic to cells in culture, are found in brain regions that show pathologic changes suggesting they contribute to CNS features of the disease (Zu et al. 2017, Neuron 95:1292-1305). Understanding the role of RAN proteins in DM2 and developing therapeutic approaches requires animal models that mirror DM2 patient disease features. Using a bacterial artificial chromosome (BAC) approach, which ensure expression in an endogenous context, our lab has successfully generated two separate lines of DM2 BAC mice from a patient-derived cell line. Both DM2 BAC mouse lines contain the entire CNBP gene with a large repeat expansion (~750 CCTG) and show expression by RT-PCR in brain and muscle tissues. We are currently characterizing these mice for DM2 molecular, histopathological and behavioral features and the potential for therapeutic applications.

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5. Structural brain abnormalities in adult-onset myotonic dystrophy type 1.

Fecto, Faisal¹, Dana Parker¹, Jacinda Sampson¹, Bryon A. Mueller², Kelvin O. Lim², Jeffrey Wozniak², Katherine A. Hagerman¹, and John W. Day¹. Affiliations: ¹Stanford University School of Medicine, Palo Alto, California, USA, ²University of Minnesota Medical School, Minneapolis, Minnesota, USA. “Structural brain abnormalities in adult-onset myotonic dystrophy type 1.”

Objective: Prior neuroimaging studies in patients with Myotonic Dystrophy Type 1 (DM1) have identified widespread morphological abnormalities (Angelini and Pinzan, 2019; Minnerop et al., 2018). The aim of this study was to determine structural brain abnormalities in patients with adult-onset DM1 and correlate them to clinical and neuropsychological measures. Methodology: Structural T1 weighted images were collected in 36 patients with DM1 and 36 age and gender matched controls. Image preprocessing and voxel-based morphometry (VBM) analysis were conducted using the Computational Anatomy Toolbox (CAT12). Global and regional gray (GM) and white matter (WM) volume differences were assessed through group comparisons and linear regression analysis with clinical and neuropsychological measures. Results: Compared to healthy controls, patients with DM1 had widespread global and regional GM and WM volume reductions that were correlated with disease severity. An enlargement of mesial temporal structures was noted in patients with DM1. GM volume loss in right inferior parietal lobule/angular gyrus and WM volume loss in left middle temporal gyrus were correlated with excessive daytime sleepiness in patients with DM1. Volume loss in the lentiform nucleus was inversely correlated with motor speed in patients with DM1. Conclusions: In this study, we describe an abnormal enlargement of mesial temporal structures, in the setting of widespread GM and WM volume loss, in patients with DM1. The GM and WM volume loss were associated with disease severity in DM1. Specific regional patterns of volume loss were associated with excessive daytime sleepiness and motor speed abnormalities in patients with DM1. References: Angelini, C., Pinzan, E., 2019. Advances in imaging of brain abnormalities in neuromuscular disease. Ther Adv Neurol Disord 12, 1756286419845567. https://doi.org/10.1177/1756286419845567 Minnerop, M., Gliem, C., Kornblum, C., 2018. Current Progress in CNS Imaging of Myotonic Dystrophy. Front Neurol 9. https://doi.org/10.3389/fneur.2018.00646

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6. Introducing a guide in physiotherapy addressing sexual function in adults with neuromuscular disorders.

Fisette-Paulhus, Isabelle¹, Mélanie Morin¹, Julie Fortin², Laura Girard-Côté^2,3, and Cynthia Gagnon^1,2. Affiliations: ¹Université de Sherbrooke, Québec, Canada, ²CIUSSS du Saguenay-Lac-St-Jean, Québec, Canada, ³Université du Québec à Chicoutimi, Saguenay, Québec, Canada. “Introducing a guide in physiotherapy addressing sexual function in adults with neuromuscular disorders.”

Physiotherapists are part of the rehabilitation team following individuals with neuromuscular disorders (NMD). Due to their impairments, people with NMD can have difficulties performing sexual activities. The practice guide has 3 main objectives: 1) To document interventions that can be applied by physiotherapists in clinical practice; 2) To propose approaches for addressing sexuality with their client; 3) To highlight issues that require expertise in pelvic rehabilitation. The guide is based on a review of the literature and the clinical experience of physiotherapists with expertise in NMD or sexuality in Quebec, Canada. It was developed using the “Rare knowledge mining methodological framework” [1]. It is recommended to introduce the topic early in therapy and follow the steps of the EX-PLISSIT model [2, 3] before offering specific suggestions. Physiotherapists should address different aspects in their assessment: interpersonal relationships, assistance required or not during sexual activities, urinary, vaginal or anorectal symptoms, erectile dysfunction, pain, fatigue and impact of the disease on sexuality. Some of the impairments or limitations that may be identified during an objective assessment can have an impact on sexual function and it is important to be aware of these issues: strength, grip, dexterity, sensibility, pain, bed mobility, motor control, etc. Interventions are presented in detail in the guide regarding physical activity, positioning, muscle weakness, fatigue, decreased mobility, erectile dysfunction, sensitivity disorders, lubrication, pain, incontinence and cardio and respiratory implications. Physiotherapists should integrate sexual function in their assessment and interventions and collaborate with other health professionals to have a multidisciplinary approach. 1. Gagnon, C. and A. Plourde, Les savoirs rares - Un défi pour une pratique fondée sur les meilleures évidences, in Conférencières invitées à l'Institut national d'excellence en santé et services sociaux (INESSS). 2014: Groupe de recherche interdisciplinaire sur les maladies neuromusculaires: Montréal. 2. Taylor, B. and S. Davis, Using the extended PLISSIT model to address sexual healthcare needs. Nurs Stand, 2006. 21(11): p. 35-40. 3. Annon, J.S., The PLISSIT Model: A Proposed Conceptual Scheme for the Behavioral Treatment of Sexual Problems. Journal of Sex Education and Therapy, 1976. 2(1): p. 1-15.

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7. Investigating subcellular RNA localization in congenital DM1 patient iPSC-derived neurons and cortical organoids.

Gosztyla, Maya L*, Kathryn H Morelli, Gene W Yeo. Affiliation: University of California San Diego, USA. “Investigating subcellular RNA localization in congenital DM1 patient iPSC-derived neurons and cortical organoids.”

Myotonic dystrophy type 1 (DM1) is caused by inherited CTG repeat expansions in the 3’-UTR of the dystrophia myotonica protein kinase (DMPK) gene. Although DM1 mostly commonly causes adult-onset muscular dystrophy, disease onset can vary from newborn to late-adult and presents a spectrum of phenotypes that correlate with CTG repeat length. When repeats exceed 2000, DM1 manifests as a congenital form (cDM1) with predominantly neurodevelopmental symptoms and mild musculoskeletal impairments. This suggests that the origin of these cognitive deficits may occur early in cortical development and persists throughout life. The mechanism by which CTG repeat expansions impact fetal cortical development to cause early-onset neurological impairment remains a fundamental unresolved question. We hypothesized that cDM1’s cognitive symptoms are caused by mislocalization of transcripts at the subcellular level. To gain insight, we used modified cellular fractionation and sequencing (CeFra-seq) in cDM1 patient iPSC-derived neurons to identify transcripts mislocalized to the nuclear, cytosolic, or insoluble cell fractions. Moving forward, we will use this data to more precisely evaluate subcellular localization using multiplexed error-robust fluorescence in situ hybridization (MERFISH) in cDM1 neurons and cerebral organoids. Together, these experiments could identify a novel mechanism of RNA dysregulation that contributes to neurological impairments in cDM1.

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8. Mechanisms of DM1 cardiac pathogenesis.

Hu, Rong-Chi* and Thomas A. Cooper. Affiliation: Baylor College of Medicine, Houston, Texas, USA. “Mechanisms of DM1 cardiac pathogenesis.”

Over half of individuals affected by Myotonic dystrophy type 1 (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 (CUGexp) transcribed from the expanded DMPK allele causes DM1 by disrupting functions of MBNL and CELF1 proteins. We are using a DM1 heart mouse model (CUG960), which expresses cardiomyocyte-specific and tetracycline-inducible RNA containing 960 interrupted CUG repeats (CUG960) to determine the degree to which loss of MBNL and gain of CELF1 activities contribute to DM1 cardiac pathogenesis using genetic approaches to test for phenotypic rescue. We delivered an adeno-associated virus serotype 9 (AAV9) for heart-specific overexpression of flag-tagged MBNL1 and myctagged MBNL2. The expression of epitope-tagged MBNLs was confirmed by Western Blotting in both left ventricles and atria. AAV9-MBNLs, but not AAV9-mCherry control, significantly reduced QRS and QTc conduction intervals in mice that were prolonged by induced CUG960. Heart weight to tibia length ratio was also significantly decreased compared to the controls. Moreover, alternative splicing events that are disrupted by CUG960 RNA were significantly rescued. These results indicate that MBNLs play a crucial role in DM1 cardiac pathogenesis. Future studies will focus on 1) determining the role of CELF1 in DM1 cardiac pathogenesis using AAV9-delivered shRNA, 2) identifying the rescue by MBNL1 and MBNL2 individually, and 3) ranking rescue by comparison to the baseline for maximum reversal when the transgene was turned off in mice taken off dox chow.

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9. Developing novel robust myotonic dystrophy brain biomarkers: A machine learning approach using diffusion tensor imaging data.

Kamali, Tahereh¹, Dana Parker¹, John W. Day¹, Jacinda Sampson¹, and Jeffery R. Wozniak². Affiliations: ¹Stanford University School of Medicine, California, USA, ²University of Minnesota, USA. “Developing novel robust myotonic dystrophy brain biomarkers: A machine learning approach using diffusion tensor imaging data.”

Numerous myotonic dystrophy (DM) patients experience neurological and cognitive effects that significantly influence their well-being. As new drug trials address the DM neurological symptoms, there is an urgent need for validated neurological biomarkers of DM. Methodology: Quantifying and understanding diffusion measures along main brain white matter fiber tracts offer a unique opportunity to reveal new insights into DM development and characterization. This work proposes advanced machine learning techniques to identify unique brain signatures of DM as a neurological biomarker, with the goal of identifying outcome measures and assessment methods for upcoming trials. The developed system is based on brain white matter tract profiles sub-band energy information and considers both long-term and short-term fluctuations caused by the disease process using wavelet approximation and detailed coefficients and utilizes a Bayesian stacked random forest to diagnose, characterize, and predict DM clinical outcomes. The evaluation data consists of fractional anisotropies calculated for twelve major white matter tracts of 96 healthy controls and 62 DM patients. Results: The proposed system discriminates DM vs. control with 86% accuracy, which is significantly higher than previous works. Additionally, it discovered DM brain biomarkers that are accurate and robust and will be helpful in planning clinical trials and monitoring clinical performance. Conclusions: The obtained results show that neurological biomarkers calculated based on the white matter Tract Profiles can accurately characterize DM1 and DM2. We are optimistic that the results of this work can accelerate pharmaceutical research and, in turn, significantly help patients with DM.

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10. Choroid plexus dysfunction and cerebral atrophy in DM1.

Kidd, Benjamin M.*, Curtis A. Nutter, Matteo Grudny, Helmut A. Carter, Marcelo Febo, and Maurice S. Swanson. Affiliations: University of Florida, College of Medicine, Gainesville, Florida, USA. “Choroid plexus dysfunction and cerebral atrophy in DM1.”

Myotonic dystrophy (DM), the most prevalent adult muscular dystrophy, is caused by microsatellite, or short tandem repeat (STR), expansions in the DMPK (DM1, CTG expansion) and CNBP (DM2, CCTG expansion) genes. While DM1 is a multisystemic disease, it is also characterized by central nervous system (CNS) symptoms, including early-onset cerebral atrophy, irregular sleep patterns, visual/spatial deficits, dysexecutive syndrome, and attentional deficits. We have recently developed Dmpk CTG expansion knockin (Dmpk CTGexp KI) mice in order to study the effects of Dmpk spatiotemporal gene expression patterns in DM1. Interestingly, the most affected CNS cell type is the epithelial cells of the choroid plexus (ChP). The ChP is located in the brain ventricles, where it produces the majority of the cerebrospinal fluid (CSF), mediates the blood-CSF barrier, regulates brain homeostasis, neuronal development, and metabolite clearance. To determine if neurogenesis pathways are perturbed in the DM1 ChP, we are analyzing ChP and CSF transcriptomes in both Mbnl2 KO and Dmpk CTGexp KI mice in conjunction with magnetic resonance imaging (MRI) to investigate if cerebral atrophy occurs in these models. We show that the telencephalic ChP (tChP) is differentially affected in our Dmpk CTGexp KI mice versus the hindbrain ChP (hChP). Furthermore, RNA splicing of TRPM3, the host gene for miR204 that regulates neurogenesis in the sub-ventricular zone (SVZ), occurs in the tChP of our mouse models. Our results suggest that loss of MBNL2 activity in the ChP due to CTGexp expression leads to early-onset neural stem cell depletion and cerebral atrophy in DM1.

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

Mishra, Subodh Kumar*, Tammy Reid, John D. Cleary, Kaalak Reddy, and J. Andrew Berglund. Affiliation: State University of New York, Albany, New York, USA. “A CTG repeat-selective screen of a natural product library reveals dietary natural compounds as potential therapeutics for myotonic dystrophy.”

Myotonic dystrophy (DM) is a multisystemic neuromuscular disease. The prevalence of DM can be as high as 1:2100 depending on the region. Expansion of a CTG and CCTG nucleotide tract in non-coding region of DMPK and CNBP gene, respectively is the responsible mutation for DM. Transcripts containing the expanded repeat form non-canonical structures that sequester the muscleblind-like (MBNL) family of splicing regulators into ribonuclear foci. This sequestration results in global aberrant splicing of MBNL pre-mRNA. Targeting expanded repeat transcription is a promising therapeutic approach for mitigating toxic RNA associated pathogenesis. Recently, we developed a DM1 HeLa cell model that permits repeat-selective screening by measuring the ratio-metric expression of r(CUG) 480 levels relative to an r(CUG) 0 control following drug treatments. In the current study, we utilized this DM1 HeLa cell model to screen a natural product small molecule library obtained from National Cancer Institute (NCI). This study revealed NP1 as a selective modulator of toxic CUG RNA abundance, as it reduces ~50% of the r(CUG)480. Further, NP1 showed significant rescue of DM associated mis-splicing events DM patient-derived myotubes and fibroblast. Several widely available foods such as red onion, grapes and broccoli are rich in NP1 and are consumed as part of the daily human diet. NP1 was also recently labeled as GRAS (Generally Recognized as Safe) by the FDA (Food and Drug Administration). This excellent safety profile with little to no adverse effects, positions NP1 as a potentially safe lead compound for therapeutic consideration in DM.

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12. Blockmir induced MBNL rescue in DM1 models.

Overby, Sarah^1*, Estefania Cerro-Herreros¹, Jorge Espinosa¹, Irene Gonzalez Martinez¹, Nerea Moreno Cervera¹, Manuel Perez-Alonzo¹, Thorleif Møller², Beatriz Llamusi³, and Ruben Artero¹. Affiliations: ¹University of Valencia, Valencia, Spain, ²Ranger Biotechnologies, Årslev, Denmark, ³ARTHEx Biotech, Valencia, Spain. “Blockmir induced MBNL rescue in DM1 models.”

The symptoms of Myotonic Dystrophy Type 1 (DM1) are multi-systemic and life-threatening. The neuromuscular disorder is rooted in a non-coding CTG microsatellite expansion in the DMPK gene that is correctly transcribed and physically sequesters the MBNL family of proteins. The high-affinity binding occurring between the proteins and the repetitions disallows MBNL proteins from performing their post-transcriptional splicing regulation leading to downstream molecular effects directly related to disease symptoms such as myotonia and muscle weakness. In this study, we build off of previously demonstrated evidence showing that the silencing of miR-23b and miR-218 can increase MBNL1 and 2 protein in DM1 cells. Here we use blockmiR antisense technology to block the binding sites of these miRNAs in order to increase MBNL translation into protein without binding to the microRNAs in DM1 muscle cells and mice. The blockmiRs show therapeutic effects with the rescue of mis-splicing, MBNL subcellular localization, and transcriptomic expression proving that this novel strategy could be a potentially viable therapy.

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13. MBNL loss of function in visceral smooth muscle as a model of myotonic dystrophy type 1.

Peterson, Janel, Geoffrey Preidis, and Thomas A. Cooper. Affiliation: Baylor College of Medicine, Houston, Texas, USA. “MBNL loss of function in visceral smooth muscle as a model of myotonic dystrophy type 1.”

Recent myotonic dystrophy type 1 (DM1) patient surveys have identified gastrointestinal (GI) disturbances as a predominant patient complaint that affects daily life and well-being. The cause of DM1 GI pathology is currently unknown and evidence supports a role for visceral smooth muscle dysfunction. The goal of this project is to elucidate the role of muscleblind-like (MBNL) loss of activity, a primary mechanism of DM1 pathology, in visceral smooth muscle function. Using mice expressing a smooth muscle specific CreERT2 and floxed Mbnl alleles (denoted as smoCRE;dHOM), I induced MBNL loss and measured gut transit time using two dye-based methods to identify changes in gut motility. I then performed RT-PCR using RNA isolated from GI smooth muscle to identify DM1-associated splicing changes. Findings demonstrated that smoCRE;dHOM mice have dysregulated GI motility and share homologous misregulated splicing events that have previously been identified in mouse MBNL KO skeletal and cardiac muscle and human DM1 tissues. These results suggest that smooth muscle specific double knockout of MBNL affects GI motility, modeling aspects of DM1 GI pathology. Mice will be used to define both smooth muscle histological and contractile changes due to MBNL loss and identify molecular mechanisms that lead to smooth muscle dysfunction by RNA sequencing and in vitro assays. This model will elucidate the myogenic basis for DM1 GI pathogenesis and provide novel insight for future investigation.

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14. Highly specific and efficient therapeutic strategy by DMPK promoter silencing in myotonic dystrophy type 1.

Porquet, Florent¹, Lin Weidong¹, Kevin Jehasse¹, Gazon Hélène¹, Silivia Blacher¹, Majeed Jamakhani¹, Laurent Massotte¹, Emmanuel Di Valentin¹, Denis Furling², Nicolas A Gillet³, Arnaud Klein^2,4, Vincent Seutin^1,4 and Luc Willems^1,4. Affiliations: ¹Université de Liège, Belgium, ²Sorbonne Université, INSERM, Institut de Myologie, France, ³Université de Namur, Belgium, ⁴These authors contributed equally to this work, in the alphabetical order. “Highly specific and efficient therapeutic strategy by DMPK promoter silencing in myotonic dystrophy type 1.”

Objective: Type 1 myotonic dystrophy (DM1) originates from an amplification of CTG microsatellites in the DMPK gene. The pathology is primarily explained by a toxic gain of function where the expanded-CUG DMPK transcripts induce, among other mechanisms, the loss of function of the MBNL proteins, triggering a wide spliceopathy. Several therapeutic strategies have been tested to neutralize the toxic DMPK transcripts or their consequences. Nevertheless, they all have at least one drawback such as lack of specificity limiting clinical applicability. Here, we investigated a new therapeutic strategy consisting of the inhibition of the DMPK promoter by a CRISPRi system in patient-derived myotubes. Methodology: Our DMPK silencing strategy by CRISPRi was tested in immortalized myoblasts from a DM1 patient or a healthy donor. Stable cell lines expressing a deactivated Cas9 conjugated to an inhibitory KRAB domain in addition to their own sgRNAs were produced by lentiviral transduction. The efficacy and specificity of our therapeutic strategy were then assessed in differentiated myotubes. Results: Our DMPK promoter inhibition strategy is highly efficient to reduce toxic DMPK transcript quantities up to 80%. This level of inhibition allows to correct the DM1 hallmark defects by reducing the presence of foci, improving the spliceopathy and normalizing an electrophysiological parameter in DM1 myotubes. Furthermore, this approach displays unprecedented high specificity as evidenced by a complete lack of off-target effects on the transcriptome from unaffected myotubes. Conclusions: We conclude that DMPK promoter inhibition is a promising strategy to be developed for DM1 treatment. Grant Support: FNRS (Belgium), Wyck Foundation, Myotonic Dystrophy Foundation, Association Belge contre les Maladies neuro-Musculaires (ABMM) and Léon Fredericq Foundation (Liège University)".

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15. How no-amplification Single-Molecule Real-time sequencing can improve the prognosis and genetic counseling in DM1 patients.

Tsai, Yuan-Chin¹, Cheryl Heiner¹, Tanya Stojkovic^2,3, Denis Furling², Guillaume Bassez^2,3, Genevieve Gourdon², and Stéphanie Tomé. Affiliations: ¹Pacific Biosciences, Menlo Park, California, USA, ²Sorbonne Université, Inserm, Institut de Myologie, Centre de Recherche en Myologie, Paris, France, ³AP-HP, centre de référence des maladies neuromusculaires Nord/Est/Ile de France, Hôpital Pitié-Salpêtrière, Paris, France. “How no-amplification Single-Molecule Real-time sequencing can improve the prognosis and genetic counseling in DM1 patients.”

Myotonic dystrophy type 1 (DM1) is the most complex and variable disorders that exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The genetic and clinical variability depend on CTG repeat number, CNG repeat interruptions and/or somatic mosaicism. Currently, none of these factors are simultaneous and accurately determined in patients due to the limitations of gold standard methods used in clinical and research laboratories. We have recently developed the amplicon targeted Single-Molecule Real-Time (SMRT) sequencing (Pacific Biosciences) in DM1 to accurately analyze the large expanded allele. However, Amplicon-based sequencing still depends on PCR and the inherent bias towards preferential amplification of smaller repeats that can be problematic in DM1. We have developed an amplification-free (No-Amp) long-read sequencing method using the CRISPR/Cas9 technology in DM1. We sequenced the DM1 locus in patients with CTG repeat expansion ranging from 130 to > 1000 CTG. The data includes long reads in the expected size range for all samples, including DM1 patients with large expanded allele. Removing PCR amplification improves the accuracy of measurement of inherited CTG repeat size and somatic repeat variations, two important key factors in the DM1 severity and age at onset. Using No-Amp, we also identified a DM1 family with an expansion composed of over 90% CCG associated with a decrease in symptom severity. No-Amp targeted SMRT sequencing represents a promising method that will overcome technical, research and diagnosis shortcomings, with translational implications to the clinic and genetic counseling in DM1.

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16. The FORCE^TM platform achieves robust knock down of toxic human nuclear DMPK RNA and foci reduction in DM1 cells and in newly developed hTfR1/DMSXL mouse model.

Zanotti, Stefano, Nelson Hsia, Timothy Weeden, Ryan J. Russo, Lydia Schlaefke, Monica Yao, Aiyun Wen, Scott Hildebrand, John Najim, Qifeng Qiu, Brendan Quinn, Mo Qatanani, Romesh Subramanian, and Oxana Beskrovnaya. Affiliation: Dyne Therapeutics, Waltham, Massachusetts, USA. “The FORCE^TM platform achieves robust knock down of toxic human nuclear DMPK RNA and foci reduction in DM1 cells and in newly developed hTfR1/DMSXL mouse model.

Therapies with antisense oligonucleotides (ASO) designed to treat myotonic dystrophy type 1 (DM1) by reducing DMPK mRNA have shown promise as potential DM1 treatments; however, their translation to the clinic has been hampered by insufficient delivery to muscle. To overcome these limitations, we developed the FORCE^TM platform by conjugating a fragment antibody (Fab) against the (hTfR)1 receptor, with ASOs targeting human DMPK mRNA. To determine the potential of FORCE to correct the DM1 phenotype in vitro, we tested their ability to knock down the human DMPK transcript, reduce nuclear foci, and correct splicing defects in myoblast cultures from several DM1 patients. The FORCE conjugate selected as our DM1 lead candidate caused over 50% DMPK mRNA knock down in patient cells, leading to nuclear foci reduction and splicing defect correction measured by BIN1 exon 11 inclusion. To enable efficacy analysis of our lead candidate against toxic human DMPK mRNA in vivo, we generated an innovative hTfR1/DMSXL mouse model that expresses the hTfR1 transgene and carries a human DMPK gene >1,000 CTG repeats. As with our DM1 patient-derived cell results, intravenous injections of our lead candidate in hTfR1/DMSXL mice resulted in a 39 - 60% reduction of the toxic human nuclear DMPK mRNA in muscle, including the heart, confirming the findings from the mRNA expression analysis. In conclusion, studies with a fully human FORCE conjugate targeting human DMPK mRNA produced encouraging data regarding the potential of the FORCE platform to be an effective therapy for patients afflicted with DM1.

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17. Small molecule GeneTACs reduce toxic nuclear foci and correct splicing defects in multiple myotonic dystrophy type 1 (DM1) cell types.

Zhang, Chengzhi, Fei Yang, Sumon Datta, Melanie Bell, James Hua, Katie Chung, Raechel Peralta, Nancy Levin, Sean Jeffries, Pratik Shah, Abhijit Bhat, and Aseem Ansari. Affiliation: Design Therapeutics, Carlsbad, California, USA. “Small molecule GeneTACs reduce toxic nuclear foci and correct splicing defects in multiple myotonic dystrophy type 1 (DM1) cell types.”

Objective: To develop a GeneTAC molecule for the treatment of myotonic dystrophy type 1 (DM1). Methodology: Gene Targeted Chimeras (GeneTACTM) are novel small molecule drug candidates designed to bind a targeted DNA sequence to modulate transcription. DM1 is an autosomal dominant monogenic progressive multisystem disease caused by CTG nucleotide repeat expansion in the 3’ non-coding region of the DMPK gene. The mutant DMPK gene transcript forms toxic nuclear foci that contain the expanded repeat and RNA-binding proteins whose sequestration is thought to lead to global splicing dysregulation and thereby cellular dysfunction. We rationally designed and synthesized multiple DM1 GeneTACs to selectively bind expanded CTG repeats and block the transcription of the mutant DMPK allele. Change in nuclear foci and correction of splicing defects in multiple DM1 cell types were evaluated by FISH and qPCR, respectively. Tissue distribution and tolerability of DM1 GeneTACs were evaluated in rodents after repeat IV administration. Results: Treatment with DM1 GeneTACs rapidly reduced the number of CUG foci in multiple DM1 patient cell types, including myoblasts and myotubes, and corrected splicing defects at low nanomolar concentrations. At well tolerated doses in rodents, DM1 GeneTAC levels in key target tissues were above those necessary to inhibit DMPK transcription and reverse splicing defects in vitro. Conclusions: The preclinical data support the potential for DM1 GeneTACs to be developed into a therapy for DM1 patients.

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