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BioDrugs : Clinical Immunotherapeutics,... Jan 2024Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic... (Comparative Study)
Comparative Study Review
Duchenne muscular dystrophy is a devastating disease that leads to progressive muscle loss and premature death. While medical management focuses mostly on symptomatic treatment, decades of research have resulted in first therapeutics able to restore the affected reading frame of dystrophin transcripts or induce synthesis of a truncated dystrophin protein from a vector, with other strategies based on gene therapy and cell signaling in preclinical or clinical development. Nevertheless, recent reports show that potentially therapeutic dystrophins can be immunogenic in patients. This raises the question of whether a dystrophin paralog, utrophin, could be a more suitable therapeutic protein. Here, we compare dystrophin and utrophin amino acid sequences and structures, combining published data with our extended in silico analyses. We then discuss these results in the context of therapeutic approaches for Duchenne muscular dystrophy. Specifically, we focus on strategies based on delivery of micro-dystrophin and micro-utrophin genes with recombinant adeno-associated viral vectors, exon skipping of the mutated dystrophin pre-mRNAs, reading through termination codons with small molecules that mask premature stop codons, dystrophin gene repair by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genetic engineering, and increasing utrophin levels. Our analyses highlight the importance of various dystrophin and utrophin domains in Duchenne muscular dystrophy treatment, providing insights into designing novel therapeutic compounds with improved efficacy and decreased immunoreactivity. While the necessary actin and β-dystroglycan binding sites are present in both proteins, important functional distinctions can be identified in these domains and some other parts of truncated dystrophins might need redesigning due to their potentially immunogenic qualities. Alternatively, therapies based on utrophins might provide a safer and more effective approach.
Topics: Humans; Dystrophin; Genetic Therapy; Muscular Dystrophy, Duchenne; Utrophin
PubMed: 37917377
DOI: 10.1007/s40259-023-00632-3 -
Stem Cell Reports Sep 2023To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However,...
To restore dystrophin protein in various mutation patterns of Duchenne muscular dystrophy (DMD), the multi-exon skipping (MES) approach has been investigated. However, only limited techniques are available to induce a large deletion to cover the target exons spread over several hundred kilobases. Here, we utilized the CRISPR-Cas3 system for MES induction and showed that dual crRNAs could induce a large deletion at the dystrophin exon 45-55 region (∼340 kb), which can be applied to various types of DMD patients. We developed a two-color SSA-based reporter system for Cas3 to enrich the genome-edited cell population and demonstrated that MES induction restored dystrophin protein in DMD-iPSCs with three distinct mutations. Whole-genome sequencing and distance analysis detected no significant off-target deletion near the putative crRNA binding sites. Altogether, dual CRISPR-Cas3 is a promising tool to induce a gigantic genomic deletion and restore dystrophin protein via MES induction.
Topics: Humans; Dystrophin; CRISPR-Cas Systems; Muscular Dystrophy, Duchenne; Binding Sites; Exons
PubMed: 37625413
DOI: 10.1016/j.stemcr.2023.07.007 -
Skeletal Muscle Nov 2023The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration...
BACKGROUND
The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice.
METHODS
The MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice.
RESULTS
MRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance.
CONCLUSIONS
These findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.
Topics: Mice; Animals; Dystrophin; Mice, Inbred mdx; Electric Impedance; Mice, Inbred C57BL; Muscular Dystrophy, Duchenne; Muscle, Skeletal; Morpholinos; Myography; Biomarkers
PubMed: 37980539
DOI: 10.1186/s13395-023-00331-1 -
Molecular Therapy : the Journal of the... Jun 2022Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young...
Gene editing has shown promise for correcting or bypassing dystrophin mutations in Duchenne muscular dystrophy (DMD). However, preclinical studies have focused on young animals with limited muscle fibrosis and wasting, thereby favoring muscle transduction, myonuclear editing, and prevention of disease progression. Here, we explore muscle-specific dystrophin gene editing following intramuscular delivery of AAV6:CK8e-CRISPR/SaCas9 in 3- and 8-year-old dystrophic CXMD dogs and provide a qualitative comparison to AAV6:CK8e-micro-dystrophin gene replacement at 6 weeks post-treatment. Gene editing restored the dystrophin reading frame in ∼1.3% of genomes and in up to 4.0% of dystrophin transcripts following excision of a 105-kb mutation containing region spanning exons 6-8. However, resulting dystrophin expression levels and effects on muscle pathology were greater with the use of micro-dystrophin gene transfer. This study demonstrates that our muscle-specific multi-exon deletion strategy can correct a frequently mutated region of the dystrophin gene in an aged large animal DMD model, but underscores that further enhancements are required to reach efficiencies comparable to AAV micro-dystrophin. Our observations also indicate that treatment efficacy and state of muscle pathology at the time of intervention are linked, suggesting the need for additional methodological optimizations related to age and disease progression to achieve relevant clinical translation of CRISPR-based therapies to all DMD patients.
Topics: Aging; Animals; CRISPR-Cas Systems; Disease Models, Animal; Disease Progression; Dogs; Dystrophin; Gene Editing; Muscle, Skeletal; Muscular Dystrophy, Duchenne
PubMed: 35143959
DOI: 10.1016/j.ymthe.2022.02.003 -
Proceedings of the National Academy of... Jan 2023Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial...
Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.
Topics: Female; Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Muscles; Gene Editing; Treatment Outcome; Mice, Inbred mdx; Muscle, Skeletal; Disease Models, Animal
PubMed: 36595689
DOI: 10.1073/pnas.2206324120 -
Molecular Neurobiology Mar 2020Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene that prevent the body-wide translation of its protein product, dystrophin. Besides a... (Review)
Review
Duchenne muscular dystrophy (DMD) is caused by frameshift mutations in the DMD gene that prevent the body-wide translation of its protein product, dystrophin. Besides a severe muscle phenotype, cognitive impairment and neuropsychiatric symptoms are prevalent. Dystrophin protein 71 (Dp71) is the major DMD gene product expressed in the brain and mutations affecting its expression are associated with the DMD neuropsychiatric syndrome. As with dystrophin in muscle, Dp71 localises to dystrophin-associated protein complexes in the brain. However, unlike in skeletal muscle; in the brain, Dp71 is alternatively spliced to produce many isoforms with differential subcellular localisations and diverse cellular functions. These include neuronal differentiation, adhesion, cell division and excitatory synapse organisation as well as nuclear functions such as nuclear scaffolding and DNA repair. In this review, we first describe brain involvement in DMD and the abnormalities observed in the DMD brain. We then review the gene expression, RNA processing and functions of Dp71. We review genotype-phenotype correlations and discuss emerging cellular/tissue evidence for the involvement of Dp71 in the neuropathophysiology of DMD. The literature suggests changes observed in the DMD brain are neurodevelopmental in origin and that their risk and severity is associated with a cumulative loss of distal DMD gene products such as Dp71. The high risk of neuropsychiatric syndromes in Duchenne patients warrants early intervention to achieve the best possible quality of life. Unravelling the function and pathophysiological significance of dystrophin in the brain has become a high research priority to inform the development of brain-targeting treatments for Duchenne.
Topics: Animals; Brain; Dystrophin; Gene Expression; Humans; Muscular Dystrophy, Duchenne; Synapses
PubMed: 31836945
DOI: 10.1007/s12035-019-01845-w -
Expert Review of Molecular Diagnostics Aug 2019: Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the... (Review)
Review
: Progressive skeletal muscle wasting is the manifesting symptom of Duchenne muscular dystrophy, an X-linked inherited disorder triggered by primary abnormalities in the gene. The almost complete loss of dystrophin isoform Dp427 causes a multi-system pathology that features in addition to skeletal muscle weakness also late-onset cardio-respiratory deficiencies, impaired metabolism and abnormalities in the central nervous system. : This review focuses on the mass spectrometry-based proteomic characterization of X-linked muscular dystrophy with special emphasis on the identification of novel biomarker candidates in skeletal muscle tissues, as well as non-muscle tissues and various biofluids. Individual sections focus on molecular and cellular aspects of the pathogenic changes in dystrophinopathy, proteomic workflows used in biomarker research, the proteomics of the dystrophin-glycoprotein complex and the potential usefulness of newly identified protein markers involved in fibre degeneration, fibrosis and inflammation. : The systematic application of large-scale proteomic surveys has identified a distinct cohort of both tissue- and biofluid-associated protein species with considerable potential for improving diagnostic, prognostic and therapy-monitoring procedures. Novel proteomic markers include components involved in fibre contraction, cellular signalling, ion homeostasis, cellular stress response, energy metabolism and the immune response, as well as maintenance of the cytoskeletal and extracellular matrix.
Topics: Biomarkers; Body Fluids; Dystrophin; Humans; Mass Spectrometry; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Protein Isoforms; Proteomics
PubMed: 31359811
DOI: 10.1080/14737159.2019.1648214 -
Cellular and Molecular Life Sciences :... Jul 2021Dystrophin is a large protein serving as local scaffolding repetitively bridging cytoskeleton and the outside of striated muscle cell. As such dystrophin is a critical... (Review)
Review
Dystrophin is a large protein serving as local scaffolding repetitively bridging cytoskeleton and the outside of striated muscle cell. As such dystrophin is a critical brick primarily in dystrophin-associated protein complex (DAGC) and in a larger submembranous unit, costamere. Accordingly, the lack of functional dystrophin laying at the root of Duchenne muscular dystrophy (DMD) drives sarcolemma instability. From this point on, the cascade inevitably leading to the death of myocyte begins. In cardiomyocytes, intracellular calcium overload and related mitochondrial-mediated cell death mainly contribute to myocardial dysfunction and dilation while other protein dysregulation and/or mislocalization may affect electrical conduction system and favor arrhythmogenesis. Although clinically DMD manifests as progressive muscle weakness and skeletal muscle symptoms define characteristic of DMD, it is the heart problem the biggest challenge that most often develop in the form of dilated cardiomyopathy (DCM). Current standards of treatment and recent progress in respiratory care, introduced in most settings in the 1990s, have improved quality of life and median life expectancy to 4th decade of patient's age. At the same time, cardiac causes of death related to DMD increases. Despite preventive and palliative cardiac treatments available, the prognoses remain poor. Direct therapeutic targeting of dystrophin deficiency is critical, however, hindered by the large size of the dystrophin cDNA and/or stochastic, often extensive genetic changes in DMD gene. The correlation between cardiac involvement and mutations affecting specific dystrophin isoforms, may provide a mutation-specific cardiac management and novel therapeutic approaches for patients with CM. Nonetheless, the successful cardiac treatment poses a big challenge and may require combined therapy to combat dystrophin deficiency and its after-effects (critical in DMD pathogenesis). This review locates the multifaceted heart problem in the course of DMD, balancing the insights into basic science, translational efforts and clinical manifestation of dystrophic heart disease.
Topics: Animals; Arrhythmias, Cardiac; Cardiomyopathies; Dystrophin; Humans; Muscular Dystrophy, Duchenne
PubMed: 34091693
DOI: 10.1007/s00018-021-03862-2 -
Gene May 2023Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic... (Review)
Review
Duchenne muscular dystrophy (DMD) is caused by the dystrophin gene mutations and is one of the most common and lethal human hereditary disorders. A novel therapeutic approach using CRISPR technology has gained attention in the treatment of DMD. Gene replacement strategies are being proposed as a promising therapeutic option to compensate the loss of function mutations. Although, the large size of the dystrophin gene and the limitations of the existing gene replacement approach, could mean the gene delivery of shortened versions of dystrophin such as midystrophin and microdystrophins. There are also other approaches: including Targeted removal of dystrophin exons to restore the reading-frame; Dual sgRNA-directed DMD exon deletion, CRISPR-SKIP strategy; reframing of dystrophin using Prime Editing technology; exon removal using twin prime technology; TransCRISTI technology to targeted exon integration into dystrophin gene. Here we provide an overview of recent progresses in dystrophin gene editing using updated versions of CRISPR to introduce novel opportunities in DMD gene therapy. Overall, the novel CRISPR based technologies are improving and expanding to allow the application of more precise gene editing for the treatment of DMD.
Topics: Humans; Muscular Dystrophy, Duchenne; Dystrophin; CRISPR-Cas Systems; Gene Editing; Genetic Therapy
PubMed: 36914142
DOI: 10.1016/j.gene.2023.147358 -
Biology Open Sep 2023Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several...
Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared the efficacies of four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e. reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.
Topics: Mice; Animals; Dystrophin; Muscular Dystrophy, Duchenne; Mice, Inbred mdx; Dependovirus; Actin Cytoskeleton; Disease Models, Animal
PubMed: 37670674
DOI: 10.1242/bio.059797