The Immunology of Takotsubo Syndrome.

Takotsubo syndrome (TTS) is a disorder characterized by transient cardiac dysfunction with ventricular regional wall motion abnormalities, primarily thought to be caused by the effects of a sudden catecholamine surge on the heart. Although the majority of patients exhibit prompt recovery of their cardiac dysfunction, TTS remains associated with increased mortality rates acutely and at long-term, and there is currently no cure for TTS. Inflammation has been shown to play a key role in determining outcomes in TTS patients, as well as in the early pathogenesis of the disorder. There are also cases of TTS patients that have been successfully treated with anti-inflammatory therapies, supporting the importance of the inflammatory response in TTS. In this article, we provide a comprehensive review of the available clinical and pre-clinical literature on the immune response in TTS, in an effort to not only better understand the pathophysiology of TTS but also to generate insights on the treatment of patients with this disorder.

DG9-conjugated morpholino rescues phenotype in SMA mice by reaching the CNS via a subcutaneous administration.

Antisense oligonucleotide-mediated (AO-mediated) therapy is a promising strategy to treat several neurological diseases, including spinal muscular atrophy (SMA). However, limited delivery to the CNS with AOs administered intravenously or subcutaneously is a major challenge. Here, we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomer (PMO) reached the CNS and significantly prolonged the median survival compared with unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited substantially higher expression of full-length survival of motor neuron 2 in both the CNS and systemic tissues compared with nontreated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localized in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.

Refining the reproducibility of a murine model of stress-induced reversible cardiomyopathy.

Despite the many advantages of isoproterenol (Iso)-induced models of cardiomyopathy, the extant literature suggests that the reproducibility of the Iso-induced stress cardiomyopathy phenotype varies considerably depending on the dose of Iso used, the mode of administration of Iso (subcutaneous vs. intraperitoneal), and the species of the animal that is being studied. Recently, we have shown that a single injection of Iso into female C57BL/6J mice provokes transient myocardial injury that is characterized by a brisk release of troponin I within 1 h, as well as a self-limited myocardial inflammatory response that is associated with increased myocardial tissue edema, inferoapical regional left ventricular (LV) wall motion abnormalities, and a transient decrease in global LV function, which were completely recovered by day 7 after the Iso injection (i.e., stress-induced reversible cardiomyopathy). Here we expand upon this initial report in this model by demonstrating important sexually dimorphic differences in the response to Iso-induced tissue injury, the ensuing myocardial inflammatory response, and changes in LV structure and function. We also provide information with respect to enhancing the reproducibility in this model by optimizing animal welfare during the procedure. The acute Iso-induced myocardial injury model provides a low-cost, relatively high-throughput small-animal model that mimics human disease (e.g., Takotsubo cardiomyopathy). Given that the model can be performed in different genetic backgrounds, as well as different experimental conditions, the acute Iso injury model should provide the cardiovascular community with a valuable nonsurgical animal model for understanding the myocardial response to tissue injury.NEW & NOTEWORTHY The present study highlights the importance of sexual dimorphism with respect to isoproterenol injury, as well as the importance of animal handling and welfare to obtain reproducible results from investigator to investigator. Based on serial observations of animal recovery (locomotor activity and grooming behavior), troponin I release, and inflammation, we identified that the method used to restrain the mice for the intraperitoneal injection was the single greatest source of variability in this model.

Current Strategies of Muscular Dystrophy Therapeutics: An Overview.

Muscular dystrophies are a group of genetic disorders characterized by varying degrees of progressive muscle weakness and degeneration. They are clinically and genetically heterogeneous but share the common histological features of dystrophic muscle. There is currently no cure for muscular dystrophies, which is of particular concern for the more disabling and/or lethal forms of the disease. Through the years, several therapies have encouragingly been developed for muscular dystrophies and include genetic, cellular, and pharmacological approaches. In this chapter, we undertake a comprehensive exploration of muscular dystrophy therapeutics under current development. Our review includes antisense therapy, CRISPR, gene replacement, cell therapy, nonsense suppression, and disease-modifying small molecule compounds.

Knocking Down DUX4 in Immortalized Facioscapulohumeral Muscular Dystrophy Patient-Derived Muscle Cells.

The third most common muscular dystrophy in the world, facioscapulohumeral muscular dystrophy (FSHD), is an inherited disorder characterized by distinct asymmetric, progressive skeletal muscle weakness that begins in the upper body and spreads to other regions with age. It is caused by mutations that induce aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is highly cytotoxic in skeletal muscle, dysregulating numerous signaling pathways as a result of its transcription factor activity. A promising set of approaches being developed to treat FSHD uses antisense oligonucleotides (AOs) to inhibit DUX4 transcript expression. Both steric-blocking and gapmer AOs have been shown to induce efficient DUX4 transcript knockdown in vitro and in vivo. Here, we describe a protocol that allows reliable screening of DUX4-targeting AOs through the evaluation of DUX4 transcript expression by quantitative real-time polymerase chain reaction. We also describe methods to assess the efficacy of these AOs by looking at their effect on the expression of DUX4 downstream target and potential off-target genes, as well as on the amelioration of in vitro muscle cell phenotypes.

Recurrent Adrenergic Stress Provokes Persistent Myocarditis in PD-1-Deficient Mice.

It is unclear how the immune system initiates effective tissue repair responses without also simultaneously activating adaptive immune responses to self-antigens released by damaged or necrotic cells. We studied the role of repetitive adrenergic mediated stress on cardiac injury wild-type and programmed death-1-deficient (PD-1-/-) mice treated with 3 intraperitoneal low doses of isoproterenol followed by an intraperitoneal injection of high-dose ISO 7 days later (ISOprimed/ISOinjury). Repetitive adrenergic stress in ISOprimed/ISOinjury PD-1-/- mice resulted in a persistent dysregulated myocardial inflammatory response characterized by the expansion of autoreactive effector CD8+ T cells, increased cardiac hypertrophy, mild left ventricular dysfunction, and increased lethality when compared with ISOprimed/ISOinjury wild-type mice.

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

Natural History of a Mouse Model Overexpressing the Dp71 Dystrophin Isoform.

Dystrophin is a 427 kDa protein that stabilizes muscle cell membranes through interactions with the cytoskeleton and various membrane-associated proteins. Loss of dystrophin as in Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle weakness and cardiac dysfunction. Multiple promoters along the dystrophin gene (DMD) give rise to a number of shorter isoforms. Of interest is Dp71, a 71 kDa isoform implicated in DMD pathology by various animal and patient studies. Strong evidence supporting such a role for Dp71, however, is lacking. Here, we use del52;WT mice to understand how Dp71 overexpression affects skeletal and cardiac muscle phenotypes. Apart from the mouse Dmd gene, del52;WT mice are heterozygous for a full-length, exon 52-deleted human DMD transgene expected to only permit Dp71 expression in muscle. Thus, del52;WT mice overexpress Dp71 through both the human and murine dystrophin genes. We observed elevated Dp71 protein in del52;WT mice, significantly higher than wild-type in the heart but not the tibialis anterior. Moreover, del52;WT mice had generally normal skeletal muscle but impaired cardiac function, exhibiting significant systolic dysfunction as early as 3 months. No histological abnormalities were found in the tibialis anterior and heart. Our results suggest that Dp71 overexpression may have more detrimental effects on the heart than on skeletal muscles, providing insight into the role of Dp71 in DMD pathogenesis.

Chromatin remodeling factor CECR2 forms tissue-specific complexes with CCAR2 and LUZP1.

Chromatin remodeling complexes alter chromatin structure to control access to DNA and therefore control cellular processes such as transcription, DNA replication, and DNA repair. CECR2 is a chromatin remodeling factor that plays an important role in neural tube closure and reproduction. Loss-of-function mutations in Cecr2 result primarily in perinatal lethal neural tube defect exencephaly, with non-penetrant mice that survive to adulthood exhibiting subfertility. CECR2 forms a complex with ISWI proteins SMARCA5 and (or) SMARCA1; however, further information on the structure and function of the complex is not known. Therefore, we identified candidate components of the CECR2-containing remodeling factor (CERF) complex in embryonic stem (ES) cells using mass spectroscopy. Both SMARCA5 and SMARCA1 were confirmed to be present in the CERF complexes in ES cells and testes. However, the novel proteins CCAR2 and LUZP1 are CERF components in ES cells, but not in the testis. This tissue specificity in mice suggests that these complexes may also have functional differences. Furthermore, LUZP1, the loss of which is also associated with exencephaly, appears to play a role in stabilizing the CERF complex in ES cells.

eSkip-Finder: a machine learning-based web application and database to identify the optimal sequences of antisense oligonucleotides for exon skipping.

Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.

Genetic Approaches for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by progressive, asymmetric muscle weakness at the face, shoulders, and upper limbs, which spreads to the lower body with age. It is the third most common inherited muscular disorder worldwide. Around 20% of patients are wheelchair-bound, and some present with extramuscular manifestations. FSHD is caused by aberrant expression of the double homeobox protein 4 (DUX4) gene in muscle. DUX4 codes for a transcription factor which, in skeletal muscle, dysregulates numerous signaling activities that culminate in cytotoxicity. Potential treatments for FSHD therefore aim to reduce the expression of DUX4 or the activity of its toxic protein product. In this article, we review how genetic approaches such as those based on oligonucleotide and genome editing technologies have been developed to achieve these goals. We also outline the challenges these therapies are facing on the road to translation, and discuss possible solutions and future directions.

A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis.

Dystrophinopathies are caused by mutations in the DMD gene. Out-of-frame deletions represent most mutational events in severe Duchenne muscular dystrophy (DMD), while in-frame deletions typically lead to milder Becker muscular dystrophy (BMD). Antisense oligonucleotide-mediated exon skipping converts an out-of-frame transcript to an in-frame one, inducing a truncated but partially functional dystrophin protein. The reading frame rule, however, has many exceptions. We thus sought to simulate clinical outcomes of exon-skipping therapies for DMD exons from clinical data of exon skip-equivalent in-frame deletions, in which the expressed quasi-dystrophins are comparable to those resulting from exon-skipping therapies. We identified a total of 1298 unique patients with exon skip-equivalent mutations in patient registries and the existing literature. We classified them into skip-equivalent deletions of each exon and statistically compared the ratio of DMD/BMD and asymptomatic individuals across the DMD gene. Our analysis identified that five exons are associated with significantly milder phenotypes than all other exons when corresponding exon skip-equivalent in-frame deletion mutations occur. Most exon skip-equivalent in-frame deletions were associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. This study indicates the importance of genotype-phenotype correlation studies in the rational design of exon-skipping therapies.

DUX4 Transcript Knockdown with Antisense 2'-O-Methoxyethyl Gapmers for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by a progressive, asymmetric weakening of muscles, starting with those in the upper body. It is caused by aberrant expression of the double homeobox protein 4 gene (DUX4) in skeletal muscle. FSHD is currently incurable. We propose to develop a therapy for FSHD using antisense 2'-O-methoxyethyl (2'-MOE) gapmers, to knock down DUX4 mRNA expression. Using immortalized patient-derived muscle cells and local intramuscular injections in the FLExDUX4 FSHD mouse model, we showed that our designed 2'-MOE gapmers significantly reduced DUX4 transcript levels in vitro and in vivo, respectively. Furthermore, in vitro, we observed significantly reduced expression of DUX4-activated downstream targets, restoration of FSHD signature genes by RNA sequencing, significant improvements in myotube morphology, and minimal off-target activity. This work facilitates the development of a promising candidate therapy for FSHD and lays down the foundation for in vivo systemic treatment studies.

Genotype-Phenotype Correlations in Duchenne and Becker Muscular Dystrophy Patients from the Canadian Neuromuscular Disease Registry.

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disorder generally caused by out-of-frame mutations in the DMD gene. In contrast, in-frame mutations usually give rise to the milder Becker muscular dystrophy (BMD). However, this reading frame rule does not always hold true. Therefore, an understanding of the relationships between genotype and phenotype is important for informing diagnosis and disease management, as well as the development of genetic therapies. Here, we evaluated genotype-phenotype correlations in DMD and BMD patients enrolled in the Canadian Neuromuscular Disease Registry from 2012 to 2019. Data from 342 DMD and 60 BMD patients with genetic test results were analyzed. The majority of patients had deletions (71%), followed by small mutations (17%) and duplications (10%); 2% had negative results. Two deletion hotspots were identified, exons 3-20 and exons 45-55, harboring 86% of deletions. Exceptions to the reading frame rule were found in 13% of patients with deletions. Surprisingly, C-terminal domain mutations were associated with decreased wheelchair use and increased forced vital capacity. Dp116 and Dp71 mutations were also linked with decreased wheelchair use, while Dp140 mutations significantly predicted cardiomyopathy. Finally, we found that 12.3% and 7% of DMD patients in the registry could be treated with FDA-approved exon 51- and 53-skipping therapies, respectively.

Detection of Locked Nucleic Acid Gapmers from Mouse Muscle Samples Using ELISA.

Antisense oligonucleotide (ASO)-mediated therapy is promising for the treatment of a variety of genetic disorders, such as Duchenne muscular dystrophy. As more ASOs advance in therapeutic development and enter clinical trials, it becomes necessary to have a means of quantifying their amounts in biological samples post-treatment. This information will be valuable for evaluating the safety and pharmacokinetic profiles of ASOs, and in deciding how the efficacy of these drugs can be improved. Gapmers are a class of ASOs characterized by having a central DNA portion that is surrounded by chemically modified nucleotides on both ends. While relatively simple and accessible methods to quantify other ASOs such as phosphorodiamidate morpholino oligomers (PMOs) using enzyme-linked immunosorbent assay (ELISA)-based techniques are available and have been used for in vivo studies, no such method is available for gapmers to our knowledge. Here, we describe a sensitive ELISA protocol that can be used to quantify the levels of locked nucleic acid (LNA) gapmers in mouse muscle tissue.

Invention and Early History of Gapmers.

Gapmers are antisense oligonucleotides composed of a central DNA segment flanked by nucleotides of modified chemistry. Hybridizing with transcripts by sequence complementarity, gapmers recruit ribonuclease H and induce target RNA degradation. Since its concept first emerged in the 1980s, much work has gone into developing gapmers for use in basic research and therapy. These include improvements in gapmer chemistry, delivery, and therapeutic safety. Gapmers have also successfully entered clinical trials for various genetic disorders, with two already approved by the U.S. Food and Drug Administration for the treatment of familial hypercholesterolemia and transthyretin amyloidosis-associated polyneuropathy. Here, we review the events surrounding the early development of gapmers, from conception to their maturity, and briefly conclude with perspectives on their use in therapy.

Cardiac Involvement in Dystrophin-Deficient Females: Current Understanding and Implications for the Treatment of Dystrophinopathies.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive condition caused primarily by out-of-frame mutations in the dystrophin gene. In males, DMD presents with progressive body-wide muscle deterioration, culminating in death as a result of cardiac or respiratory failure. A milder form of DMD exists, called Becker muscular dystrophy (BMD), which is typically caused by in-frame dystrophin gene mutations. It should be emphasized that DMD and BMD are not exclusive to males, as some female dystrophin mutation carriers do present with similar symptoms, generally at reduced levels of severity. Cardiac involvement in particular is a pressing concern among manifesting females, as it may develop into serious heart failure or could predispose them to certain risks during pregnancy or daily life activities. It is known that about 8% of carriers present with dilated cardiomyopathy, though it may vary from 0% to 16.7%, depending on if the carrier is classified as having DMD or BMD. Understanding the genetic and molecular mechanisms underlying cardiac manifestations in dystrophin-deficient females is therefore of critical importance. In this article, we review available information from the literature on this subject, as well as discuss the implications of female carrier studies on the development of therapies aiming to increase dystrophin levels in the heart.

Inhibition of DUX4 expression with antisense LNA gapmers as a therapy for facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD), characterized by progressive muscle weakness and deterioration, is genetically linked to aberrant expression of DUX4 in muscle. DUX4, in its full-length form, is cytotoxic in nongermline tissues. Here, we designed locked nucleic acid (LNA) gapmer antisense oligonucleotides (AOs) to knock down DUX4 in immortalized FSHD myoblasts and the FLExDUX4 FSHD mouse model. Using a screening method capable of reliably evaluating the knockdown efficiency of LNA gapmers against endogenous DUX4 messenger RNA in vitro, we demonstrate that several designed LNA gapmers selectively and effectively reduced DUX4 expression with nearly complete knockdown. We also found potential functional benefits of AOs on muscle fusion and structure in vitro. Finally, we show that one of the LNA gapmers was taken up and induced effective silencing of DUX4 upon local treatment in vivo. The LNA gapmers developed here will help facilitate the development of FSHD therapies.