Changes in Myonuclear Number During Postnatal Growth – Implications for AAV Gene Therapy for Muscular Dystrophy

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration. AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for microdystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies

Generation of two genomic-integration-free DMD iPSC lines with mutations affecting all dystrophin isoforms and potentially amenable to exon-skipping

Duchenne muscular dystrophy (DMD) is the most common paediatric muscular dystrophy and is caused by mutations in the DYSTROPHIN gene. We generated two induced pluripotent stem cell (iPSC) lines from DMD patients with nonsense mutations in exons 68 (UCLi011-A) or 70 (UCLi012-A) by transfecting reprogramming mRNAs. Both mutations affect expression of all dystrophin isoforms. iPSCs expressed pluripotency-associated markers, differentiated into cells of the three germ layers in vitro and had normal karyotypes. The selected mutations are potentially amenable to read-through therapies, exon-skipping and gene-editing. These new iPSCs are also relevant to study DYSTROPHIN role in tissues other than skeletal muscle

Defining and Identifying Satellite Cell-opathies within Muscular Dystrophies and Myopathies

Muscular dystrophies and congenital myopathies arise from specific genetic mutations causing skeletal muscle weakness that reduces quality of life. Muscle health relies on resident muscle stem cells called satellite cells, which enable life-course muscle growth, maintenance, repair and regeneration. Such tuned plasticity gradually diminishes in muscle diseases, suggesting compromised satellite cell function. A central issue however, is whether the pathogenic mutation perturbs satellite cell function directly and/or indirectly via an increasingly hostile microenvironment as disease progresses. Here, we explore the effects on satellite cell function of pathogenic mutations in genes (myopathogenes) that associate with muscle disorders, to evaluate muscle pathological hallmarks that define dysfunctional satellite cells. We deploy transcriptomic analysis and comparison between muscular dystrophies and myopathies to determine the contribution of satellite cell dysfunction using literature, expression dynamics of myopathogenes and correlation with expression of the satellite cell marker PAX7. Our multimodal approach extends current pathological classifications to define Satellite Cell-opathies: muscle disorders in which satellite cell dysfunction contributes to pathology. Primary Satellite Cell-opathies are conditions where mutations in a myopathogene directly affect satellite cell function, such as in Progressive Congenital Myopathy with Scoliosis (MYOSCO) and Carey-Fineman-Ziter Syndrome (CFZS). Primary satellite cell-opathies are generally characterised as being congenital with general hypotonia, and specific involvement of respiratory, trunk and facial muscles, although serum CK levels are usually within the normal range. Secondary Satellite Cell-opathies have mutations in myopathogene that affect both satellite cells and muscle fibres. Such classification aids diagnosis and predicting probable disease course, as well as informing on treatment and therapeutic development

Transgenic Overexpression of LARGE Induces alpha-Dystroglycan Hyperglycosylation in Skeletal and Cardiac Muscle

Background: LARGE is one of seven putative or demonstrated glycosyltransferase enzymes defective in a common group of muscular dystrophies with reduced glycosylation of alpha-dystroglycan. Overexpression of LARGE induces hyperglycosylation of alpha-dystroglycan in both wild type and in cells from dystroglycanopathy patients, irrespective of their primary gene defect, restoring functional glycosylation. Viral delivery of LARGE to skeletal muscle in animal models of dystroglycanopathy has identical effects in vivo, suggesting that the restoration of functional glycosylation could have therapeutic applications in these disorders. Pharmacological strategies to upregulate Large expression are also being explored.Methodology/Principal Findings: In order to asses the safety and efficacy of long term LARGE over-expression in vivo, we have generated four mouse lines expressing a human LARGE transgene. On observation, LARGE transgenic mice were indistinguishable from the wild type littermates. Tissue analysis from young mice of all four lines showed a variable pattern of transgene expression: highest in skeletal and cardiac muscles, and lower in brain, kidney and liver. Transgene expression in striated muscles correlated with alpha-dystroglycan hyperglycosylation, as determined by immunoreactivity to antibody IIH6 and increased laminin binding on an overlay assay. Other components of the dystroglycan complex and extracellular matrix ligands were normally expressed, and general muscle histology was indistinguishable from wild type controls. Further detailed muscle physiological analysis demonstrated a loss of force in response to eccentric exercise in the older, but not in the younger mice, suggesting this deficit developed over time. However this remained a subclinical feature as no pathology was observed in older mice in any muscles including the diaphragm, which is sensitive to mechanical load-induced damage.Conclusions/Significance: This work shows that potential therapies in the dystroglycanopathies based on LARGE upregulation and alpha-dystroglycan hyperglycosylation in muscle should be safe

Genome Editing and Muscle Stem Cells as a Therapeutic Tool for Muscular Dystrophies

PURPOSE OF REVIEW: Muscular dystrophies are a group of severe degenerative disorders characterized by muscle fiber degeneration and death. Therapies designed to restore muscle homeostasis and to replace dying fibers are being experimented, but none of those in clinical trials are suitable to permanently address individual gene mutation. The purpose of this review is to discuss genome editing tools such as CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated), which enable direct sequence alteration and could potentially be adopted to correct the genetic defect leading to muscle impairment. RECENT FINDINGS: Recent findings show that advances in gene therapy, when combined with traditional viral vector-based approaches, are bringing the field of regenerative medicine closer to precision-based medicine. SUMMARY: The use of such programmable nucleases is proving beneficial for the creation of more accurate in vitro and in vivo disease models. Several gene and cell-therapy studies have been performed on satellite cells, the primary skeletal muscle stem cells involved in muscle regeneration. However, these have mainly been based on artificial replacement or augmentation of the missing protein. Satellite cells are a particularly appealing target to address these innovative technologies for the treatment of muscular dystrophies

Gapmer Antisense Oligonucleotides Suppress the Mutant Allele of COL6A3 and Restore Functional Protein in Ullrich Muscular Dystrophy

Dominant-negative mutations in the genes that encode the three major a chains of collagen type VI, COL6A1, COL6A2, and COL6A3, account for more than 50% of Ullrich congenital muscular dystrophy patients and nearly all Bethlem myopathy patients. Gapmer antisense oligonucleotides (AONs) are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In this study, we exploited the application of the allele-specific silencing approach by gapmer AON as a potential therapy for Collagen-VI-related congenital muscular dystrophy (COL6-CMD). A series of AONs were designed to selectively target an 18-nt heterozygous genomic deletion in exon 15 of COL6A3 at the mRNA and pre-mRNA level. We showed that gapmer AONs can selectively suppress the expression of mutant transcripts at both pre-mRNA and mRNA levels, and that the latter strategy had a far stronger efficiency than the former. More importantly, we found that silencing of the mutant transcripts by gapmer AONs increased the deposition of collagen VI protein into the extracellular matrix, thus restoring functional protein production. Our findings provide a clear proof of concept for AON allele-specific silencing as a therapeutic approach for COL6-CM

Genetic therapies for inherited neuromuscular disorders

Inherited neuromuscular disorders encompass a broad group of genetic conditions, and the discovery of these underlying genes has expanded greatly in the past three decades. The discovery of such genes has enabled more precise diagnosis of these disorders and the development of specific therapeutic approaches that target the genetic basis and pathophysiological pathways. Such translational research has led to the approval of two genetic therapies by the US Food and Drug Administration: eteplirsen for Duchenne muscular dystrophy and nusinersen for spinal muscular atrophy, which are both antisense oligonucleotides that modify pre-mRNA splicing. In this Review we aim to discuss new genetic therapies and ongoing clinical trials for Duchenne muscular dystrophy, spinal muscular atrophy, and other less common childhood neuromuscular disorders

Microwave heating improvement: permittivity characterization of water–ethanol and water–NaCl binary mixtures

Microwave heating offers a lot of advantages compared to conventional heating methods in the chemical reactions field due to its positive effects on reaction time and selectivity. Dielectric properties, and in particular permittivity, of substances and mixtures, are important for the optimization of microwave heating processes; notwithstanding this, specific databases are poor and far from being complete, and in the scientific literature very little data regarding these properties can be found. In this work, impedance measurements were carried out using a specially designed system to get the real and imaginary parts of the dielectric constant. The apparatus was tested in the estimation of permittivity of water–ethanol and water–NaCl mixtures, varying their composition to obtain a wide range of permittivity values. The results were compared to literature data and fitted with available literature models to verify the correspondence between them, finding that permittivity dependence on mixture composition can be effectively described by the models