scholarly journals Intramuscular Evaluation of Chimeric Locked Nucleic Acid/2’OMethyl-Modified Antisense Oligonucleotides for Targeted Exon 23 Skipping in Mdx Mice

2021 ◽  
Vol 14 (11) ◽  
pp. 1113
Author(s):  
Michaella Georgiadou ◽  
Melina Christou ◽  
Kleitos Sokratous Sokratous ◽  
Jesper Wengel ◽  
Kyriaki Michailidou ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Therapeutic Potential ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Locked Nucleic Acid ◽  
Mdx Mice ◽  
Muscle Fibres ◽  
Systemic Delivery ◽  
Reading Frame ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is a fatal disorder characterised by progressive muscle wasting. It is caused by mutations in the dystrophin gene, which disrupt the open reading frame leading to the loss of functional dystrophin protein in muscle fibres. Antisense oligonucleotide (AON)-mediated skipping of the mutated exon, which allows production of a truncated but partially functional dystrophin protein, has been at the forefront of DMD therapeutic research for over two decades. Nonetheless, novel nucleic acid modifications and AON designs are continuously being developed to improve the clinical benefit profile of current drugs in the DMD pipeline. We herein designed a series of 15mer and 20mer AONs, consisting of 2’O-Methyl (2’OMe)- and locked nucleic acid (LNA)-modified nucleotides in different percentage compositions, and assessed their efficiency in inducing exon 23 skipping and dystrophin restoration in locally injected muscles of mdx mice. We demonstrate that LNA/2’OMe AONs with a 30% LNA composition were significantly more potent in inducing exon skipping and dystrophin restoration in treated mdx muscles, compared to a previously tested 2’OMe AON and LNA/2’OMe chimeras with lower or higher LNA compositions. These results underscore the therapeutic potential of LNA/2’OMe AONs, paving the way for further experimentation to evaluate their benefit-toxicity profile following systemic delivery.

scholarly journals Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

2017 ◽  
Vol 9 (418) ◽  
pp. eaan8081 ◽  
Author(s):  
Leonela Amoasii ◽  
Chengzu Long ◽  
Hui Li ◽  
Alex A. Mireault ◽  
John M. Shelton ◽  
...  
Keyword(s):  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Neuromuscular Disorders ◽  
Dystrophin Gene ◽  
Muscle Disease ◽  
Systemic Delivery ◽  
Specific Expression ◽  
Reading Frame ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

scholarly journals Structural Perturbations of Exon Skipping Edits within the Dystrophin D20:24 Region

2020 ◽  
Author(s):  
Xin Niu ◽  
Nick Menhart
Keyword(s):  
Protein Expression ◽  
Genetic Defect ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Protein Domain ◽  
Reading Frame ◽  
Structural Perturbations ◽  
Protein Domain Structure ◽  
And Function ◽  
Dystrophin Protein

AbstractExon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from inclusion in the mature mRNA. Of course, this also results in loss of the corresponding region from the cognate protein, which is one possible therapeutic aim. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame in the dystrophin gene, creating a frameshift and abolishing protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is the restoration of the reading frame by skipping an exon adjacent to the patient’s original defect. However, the therapeutically expressed protein is of course edited, and missing both the region of the underlying genetic defect, as well as the therapeutically skipped exon. While restoring some protein expression is good, how removing some region from the middle of a protein effects its structure and function is unclear. Complicating this in the case of DMD is the fact that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are known, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. What has been unclear is whether and how exon editing can be done to minimize these perturbations. In this study we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein, and identify for the first time, exon edits that appear to maintain structural stability similar to wildtype protein. We also identify factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the underlying exon structure interacts with the protein domain structure.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice

2019 ◽  
Author(s):  
Ngoc Lu-Nguyen ◽  
Arnaud Ferry ◽  
Frederick J Schnell ◽  
Gunnar J Hanson ◽  
Linda Popplewell ◽  
...  
Keyword(s):  
Combined Therapy ◽  
Muscle Growth ◽  
Exon Skipping ◽  
Negative Regulator ◽  
Mdx Mice ◽  
Reading Frame ◽  
Combination Approach ◽  
Dmd Gene ◽  
The One ◽  
Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500–5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

scholarly journals Evaluating the potential of novel genetic approaches for the treatment of Duchenne muscular dystrophy

2021 ◽  
Author(s):  
Vratko Himič ◽  
Kay E. Davies
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Structural Integrity ◽  
Viral Vector ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Pharmacological Treatments ◽  
Genetic Sequencing ◽  
Reading Frame ◽  
Target Effects

AbstractDuchenne muscular dystrophy (DMD) is an X-linked progressive muscle-wasting disorder that is caused by a lack of functional dystrophin, a cytoplasmic protein necessary for the structural integrity of muscle. As variants in the dystrophin gene lead to a disruption of the reading frame, pharmacological treatments have only limited efficacy; there is currently no effective therapy and consequently, a significant unmet clinical need for DMD. Recently, novel genetic approaches have shown real promise in treating DMD, with advancements in the efficacy and tropism of exon skipping and surrogate gene therapy. CRISPR-Cas9 has the potential to be a ‘one-hit’ curative treatment in the coming decade. The current limitations of gene editing, such as off-target effects and immunogenicity, are in fact partly constraints of the delivery method itself, and thus research focus has shifted to improving the viral vector. In order to halt the loss of ambulation, early diagnosis and treatment will be pivotal. In an era where genetic sequencing is increasingly utilised in the clinic, genetic therapies will play a progressively central role in DMD therapy. This review delineates the relative merits of cutting-edge genetic approaches, as well as the challenges that still need to be overcome before they become clinically viable.

Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice

2002 ◽  
Vol 283 (3) ◽  
pp. C773-C784 ◽  
Author(s):  
Karl Rouger ◽  
Martine Le Cunff ◽  
Marja Steenman ◽  
Marie-Claude Potier ◽  
Nathalie Gibelin ◽  
...  
Keyword(s):  
Dystrophin Gene ◽  
Diaphragm Muscle ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
First Year ◽  
Temporal Gene Expression ◽  
Cell Functions ◽  
Hindlimb Muscles ◽  
Genes Encoding ◽  
Dystrophin Protein

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.

scholarly journals Autophagy in the heart is enhanced and independent of disease progression in mus musculus dystrophinopathy models

2019 ◽  
Vol 8 ◽  
pp. 204800401987958
Author(s):  
HR Spaulding ◽  
C Ballmann ◽  
JC Quindry ◽  
MB Hudson ◽  
JT Selsby
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Disease Progression ◽  
Gene Mutations ◽  
Dystrophin Gene ◽  
Mdx Mice ◽  
Dystrophin Deficiency ◽  
Muscle Wasting Disease ◽  
Dystrophin Protein

Background Duchenne muscular dystrophy is a muscle wasting disease caused by dystrophin gene mutations resulting in dysfunctional dystrophin protein. Autophagy, a proteolytic process, is impaired in dystrophic skeletal muscle though little is known about the effect of dystrophin deficiency on autophagy in cardiac muscle. We hypothesized that with disease progression autophagy would become increasingly dysfunctional based upon indirect autophagic markers. Methods Markers of autophagy were measured by western blot in 7-week-old and 17-month-old control (C57) and dystrophic (mdx) hearts. Results Counter to our hypothesis, markers of autophagy were similar between groups. Given these surprising results, two independent experiments were conducted using 14-month-old mdx mice or 10-month-old mdx/Utrn± mice, a more severe model of Duchenne muscular dystrophy. Data from these animals suggest increased autophagosome degradation. Conclusion Together these data suggest that autophagy is not impaired in the dystrophic myocardium as it is in dystrophic skeletal muscle and that disease progression and related injury is independent of autophagic dysfunction.

scholarly journals The Interplay of Mitophagy and Inflammation in Duchenne Muscular Dystrophy

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 648
Author(s):  
Andrea L. Reid ◽  
Matthew S. Alexander
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mitochondrial Dysfunction ◽  
Disease Onset ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Muscle Disease ◽  
Mitochondrial Morphology ◽  
Gene Therapies ◽  
Dystrophin Protein

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

scholarly journals Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice

2015 ◽  
Vol 23 (8) ◽  
pp. 1341-1348 ◽  
Author(s):  
Ngoc B Lu-Nguyen ◽  
Susan A Jarmin ◽  
Amer F Saleh ◽  
Linda Popplewell ◽  
Michael J Gait ◽  
...  
Keyword(s):  
Exon Skipping ◽  
Open Reading Frame ◽  
Mdx Mice ◽  
Reading Frame

Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy

2018 ◽  
Vol 10 (444) ◽  
pp. eaat0195 ◽  
Author(s):  
Xianjun Gao ◽  
Ning Ran ◽  
Xue Dong ◽  
Bingfeng Zuo ◽  
Rong Yang ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Surface Protein ◽  
Functional Improvement ◽  
Therapeutic Drug ◽  
Mdx Mice ◽  
Dystrophin Expression ◽  
Targeting Peptide ◽  
Dystrophin Protein ◽  
Diagnostics And Therapy

Exosomes are circulating nanovesicular carriers of macromolecules, increasingly used for diagnostics and therapeutics. The ability to load and target patient-derived exosomes without altering exosomal surfaces is key to unlocking their therapeutic potential. We demonstrate that a peptide (CP05) identified by phage display enables targeting, cargo loading, and capture of exosomes from diverse origins, including patient-derived exosomes, through binding to CD63—an exosomal surface protein. Systemic administration of exosomes loaded with CP05-modified, dystrophin splice–correcting phosphorodiamidate morpholino oligomer (EXOPMO) increased dystrophin protein 18-fold in quadriceps of dystrophin-deficient mdx mice compared to CP05-PMO. Loading CP05-muscle–targeting peptide on EXOPMO further increased dystrophin expression in muscle with functional improvement without any detectable toxicity. Our study demonstrates that an exosomal anchor peptide enables direct, effective functionalization and capture of exosomes, thus providing a tool for exosome engineering, probing gene function in vivo, and targeted therapeutic drug delivery.

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