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Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle. [Copyright &y& Elsevier]

Myotonic dystrophy (DM1) is caused by the expansion of a trinucleotide repeat (CTG) located in the 3'untranslated region of the myotonic dystrophy protein kinase gene, for which currently there is no effective treatment. The data available suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis. This indicates that the specific targeting of the mutant DMPK transcripts is essential to raise the rationale basis for the development of a specific gene therapy for DM1. We have produced a retrovirus which expresses a 149-bp antisense RNA complementary to the (CUG)13 repeats and to the 110-bp region following the repeats sequence to increase the specificity. This construct was introduced into human DM1 myoblasts, resulting in a preferential decrease in mutant DMPK transcripts, and effective restoration of human DM1 myoblast functions such as myoblast fusion and the uptake of glucose. It was previously shown that delay of muscle differentiation and insulin resistance in DM1 are associated with misregulation of CUGBP1 protein levels. The analysis of CUGBP1 levels and activity in DM1 cells expressing the antisense RNA indicated a correction of CUGBP1 expression in infected DM1 cells. We therefore show that current antisense RNA delivered in vitro using a retrovirus is not only capable of inhibiting mutant DMPK transcripts, but also can ameliorate dystrophic muscle pathology at the cellular levels.Gene Therapy (2003) 10, 795-802. doi:10.1038/sj.gt.3301955 [ABSTRACT FROM AUTHOR]

PepGen's Enhanced Delivery Oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and nuclear uptake of therapeutic oligonucleotides. PGN-EDODM1, in clinical trials for the treatment of myotonic dystrophy type 1 (DM1), is designed to bind pathogenic CUG repeat expansions in DMPK mRNA and liberate MBNL1 protein without degrading DMPK transcripts. Liberation of sequestered MBNL1 is expected to restore the normal splicing of multiple downstream transcripts; a central cause of DM1 pathology. Indeed, in vitro treatment of DM1 myotubes showed PGN-EDODM1 delivery to the nucleus, and treatment of DM1 patient myotubes with both low-CUG and high-CUG repeats demonstrated similar dose-dependent reduction of pathogenic myonuclear foci, liberation of MBNL1 from foci, and correction of mis-splicing without DMPK RNA degradation. A single intravenous (IV) dose of PGN-EDODM1 to HSALR mice resulted in dose-dependent correction of mis-splicing and improvement in myotonia. Following repeat-dosing of PGN-EDODM1 once every 4 weeks, near complete resolution of DM1 pathology including correction of mis-splicing and resolution of myotonia was observed. Toxicology studies in NHPs indicate repeat dosing with PGN-EDODM1 was generally safe and well tolerated. No persistent elevation of kidney biomarkers was observed at doses through 60 mg/kg. Additionally, there were no adverse findings in the kidney after 4 monthly doses of 60 mg/kg, nor notable hematologic or hepatic or cardiovascular effects. Currently, there are no approved therapies for DM1. Nonclinical pharmacology and safety studies with PGN-EDODM1 showed meaningful therapeutic potential on splicing correction and myotonia correction and support the ongoing Phase 1 single-ascending dose study FREEDOM-DM1 in adults with DM1. [ABSTRACT FROM AUTHOR]

Abstract: Expansion of repeated sequences in non-coding regions of different genes causes a number of inherited diseases including myotonic dystrophies, Huntington disease-like 2, Fragile X tremor/ataxia syndrome and spinocerebellar ataxia 8, 10, 12, 31. Involvement of an RNA gain-of-function mechanism in pathological case has been described and studied in-depth in myotonic dystrophy type 1 (DM1). This inherited neuromuscular disorder is caused by a (CTG)n >50 expansion in the 3′ non-coding region of the dystrophia myotonica-protein kinase (DMPK) gene. Expanded CUG transcripts (CUGexp-RNAs) are sequestered in the nucleus within small aggregates and interfere with the regulatory splicing activities of MBNL1 and CELF1 RNA-binding proteins, leading to the misregulation of the alternative splicing of several transcripts. Despite the relevance of aberrant splicing events in this complex pathology, the CUGexp-RNAs trans-dominant effects alter other splicing-independent processes that may also contribute to DM1 pathogenesis. This review will focus on toxic RNA gain-of-function as a pathologic mechanism for DM1 and other repeat expansion disorders. [Copyright &y& Elsevier]

Abstract: Introduction. – In this study, we tested the ability of IGF-1 (insulin-like growth factor-1) to increase the size of human myotubes in culture. Synthèse des faits. – IGF-1 treatment induces hypertrophy of human myotubes characterized by a dose dependant increase in the mean number of nuclei per myotube, in the size of the myotubes and a significant increase of 96,4±50,48% (P =0,002) of the ratio myosin/émerine. Conclusion. – In this study, we showed that IGF-1 induces an increase in the size of human myotubes in culture and increases myosin synthesis, thus mimicking the hypertrophy described in vivo. This model will be used for further analysis of the molecular mechanisms involved in this hypertrophy. [Copyright &y& Elsevier]

PepGen's enhanced delivery oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDODM1 is being evaluated for the treatment of myotonic dystrophy type 1 (DM1). PGN-EDODM1 binds to toxic CUG repeat expansion (CUGexp) in DMPK mRNA and acts to liberate sequestered MBNL1 protein without degrading DMPK transcript. Liberation of MBNL1 is hypothesized to restore splicing profiles of multiple downstream transcripts; a central cause of DM1 pathology. PGN-EDODM1 pharmacology was characterized using human immortalized myoblasts and the HSALR transgenic mouse model of DM1, and non-human primates (NHPs) were used to evaluate DMPK levels. Control and DM1 myoblasts (∼2600 CTGexp) were differentiated and treated with PGN EDODM1, which resulted in dose-dependent reduction in pathogenic myonuclear foci, liberation of MBNL1 from foci, and correction of mis-splicing in DM1 cells, while DMPK levels remained unchanged. Single intravenous (IV) dose of PGN-EDODM1 or control administered to HSALR mice resulted in high muscle concentrations of PGN-EDODM1, resolution of myotonia, dose-dependent correction of mis-splicing, and no significant impact on HSA expression when evaluated 2-weeks postdose. Splicing correction in HSALR mice persisted up to 24 weeks. Additional data from a repeat-dose study in HSALR mice with low PGN EDODM1 doses will be presented. Repeat IV doses of PGN-EDODM1 or control administered to NHPs every 2 weeks (10, 30, or 60mg/kg) or every 4 weeks (60mg/kg) did not result in decreases in DMPK transcript levels when evaluated 1 week after the final dose. Currently, there are no approved therapies for DM1. Nonclinical pharmacology studies with PGN-EDODM1 showed considerable therapeutic potential. Nonclinical data support the Phase 1 single-ascending dose study in adults with DM1. [ABSTRACT FROM AUTHOR]

Voltage-gated calcium channels (CaVs or VGCCs) are transmembrane ion channel proteins acting as major regulators of calcium-related cellular functions. In skeletal muscle, the essential component of the pore channel is the CaVα1 subunit, which displays the voltage sensor activity responding to membrane depolarization following electrical activation. Though, the fine-tuning of Ca2+-dependent signals is guaranteed by the modulatory roles of the CaV auxiliary subunits β, α2δ, and γ. The CaVβ proteins exist as several splice variants: in skeletal muscle, during embryogenesis the isoform mainly expressed is CaVβ1E, while in adult muscle CaVβ1D is the constitutive isoform. Our team recently demonstrated that, in adult muscle, the embryonic CaVβ1E boosts downstream Growth Differentiation Factor 5 (GDF5) signalling after nerve damage to counteract muscle wasting, therefore playing a crucial role in adult muscle mass homeostasis when electrical activity is impaired. The increase in CaVβ1E expression is secondary to an alternative first exon splicing of the Cacnb1 gene. However, the mechanisms leading to this splicing event are unknown to date. To decipher these mechanisms, we first analyzed RNA-seq data on innervated versus denervated adult mouse muscle transcripts and showed that, among all identified splicing factors, MuscleBlind-Like protein (Mbnl)-1 is under-expressed while Mbnl3 is the most strongly over-expressed after denervation. Moreover, recent publications have shown a mis-splicing of CACNB1 in a human cellular model of Myotonic dystrophy (DM) in which Mbnl1 is sequestrated in nuclear aggregates. Importantly, our preliminary data show an increased Cacnb1E expression in several mouse models of DM1. In addition, we showed that Mbnl3 expression follows the same kinetics as Cacnb1E in innervated and denervated adult muscles in mice. Overall, these data suggest MBNLs as potential candidates regulating Cacnb1 expression. We are now assessing, in vitro , the effects of Mbnls protein modulation on Cacnb1 expression, by evaluating their role on exon splicing but also on the preferential use of specific promoters and their eventual effects on alternative polyadenylation affecting Cacnb1 transcript stability. [ABSTRACT FROM AUTHOR]

Aims: Myotonic dystrophy type 1 (DM1), one of the most common forms of inherited neuromuscular disorders in the adult, is characterized by progressive muscle weakness and wasting leading to distal muscle atrophy whereas proximal muscles of the same patients are spared during the early phase of the disease. In this report, the role of satellite cell dysfunction in the progressive muscular atrophy has been investigated. Methods: Biopsies were obtained from distal and proximal muscles of the same DM1 patients. Histological and immunohistological analyses were carried out and the past regenerative history of the muscle was evaluated. Satellite cell number was quantified in vivo and proliferative capacity was determined in vitro. Results: The size of the CTG expansion was positively correlated with the severity of the symptoms and the degree of muscle histopathology. Marked atrophy associated with typical DM1 features was observed in distal muscles of severely affected patients whereas proximal muscles were relatively spared. The number of satellite cells was significantly increased (twofold) in the distal muscles whereas very little regeneration was observed as confirmed by telomere analyses and developmental MyHC staining (0.3–3%). The satellite cells isolated from the DM1 distal muscles had a reduced proliferative capacity (36%) and stopped growing prematurely with telomeres longer than control cells (8.4 vs. 7.1 kb), indicating that the behaviour of these precursor cells was modified. Conclusions: Our results indicate that alterations in the basic functions of the satellite cells progressively impair the muscle mass maintenance and/or regeneration resulting in gradual muscular atrophy. [ABSTRACT FROM AUTHOR]

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo–Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease. [ABSTRACT FROM AUTHOR]