Novel long noncoding RNAs (lncRNAs) in myogenesis: a miR-31 overlapping lncRNA transcript controls myoblast differentiation.

Transcriptome analysis allowed the identification of new long noncoding RNAs differentially expressed during murine myoblast differentiation. These transcripts were classified on the basis of their expression under proliferating versus differentiated conditions, muscle-restricted activation, and subcellular localization. Several species displayed preferential expression in dystrophic (mdx) versus wild-type muscles, indicating their possible link with regenerative processes. One of the identified transcripts, lnc-31, even if originating from the same nuclear precursor of miR-31, is produced by a pathway mutually exclusive. We show that lnc-31 and its human homologue hsa-lnc-31 are expressed in proliferating myoblasts, where they counteract differentiation. In line with this, both species are more abundant in mdx muscles and in human Duchenne muscular dystrophy (DMD) myoblasts, than in their normal counterparts. Altogether, these data suggest a crucial role for lnc-31 in controlling the differentiation commitment of precursor myoblasts and indicate that its function is maintained in evolution despite the poor sequence conservation with the human counterpart.

Exon 45 skipping through U1-snRNA antisense molecules recovers the Dys-nNOS pathway and muscle differentiation in human DMD myoblasts.

Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.

miRNAs as serum biomarkers for Duchenne muscular dystrophy.

Dystrophin absence in Duchenne muscular dystrophy (DMD) causes severe muscle degeneration. We describe that, as consequence of fibre damage, specific muscle-miRNAs are released in to the bloodstream of DMD patients and their levels correlate with the severity of the disease. The same miRNAs are abundant also in the blood of mdx mice and recover to wild-type levels in animals 'cured' through exon skipping. Even though creatine kinase (CK) blood levels have been utilized as diagnostic markers of several neuromuscular diseases, including DMD, we demonstrate that they correlate less well with the disease severity. Although the analysis of a larger number of patients should allow to obtain more refined correlations with the different stages of disease progression, we propose that miR-1, miR-133, and miR-206 are new and valuable biomarkers for the diagnosis of DMD and possibly also for monitoring the outcomes of therapeutic interventions in humans. Despite many different DMD therapeutic approaches are now entering clinical trials, a unifying method for assessing the benefit of different treatments is still lacking.

miR-31 modulates dystrophin expression: new implications for Duchenne muscular dystrophy therapy.

Duchenne muscular dystrophy (DMD)--which is caused by mutations in the dystrophin gene-is one of the most severe myopathies. Among therapeutic strategies, exon skipping allows the rescue of dystrophin synthesis through the production of a shorter but functional messenger RNA. Here, we report the identification of a microRNA--miR-31--that represses dystrophin expression by targeting its 3' untranslated region. In human DMD myoblasts treated with exon skipping, we demonstrate that miR-31 inhibition increases dystrophin rescue. These results indicate that interfering with miR-31 activity can provide an ameliorating strategy for those DMD therapies that are aimed at efficiently recovering dystrophin synthesis.

Exon skipping and duchenne muscular dystrophy therapy: selection of the most active U1 snRNA antisense able to induce dystrophin exon 51 skipping.

One promising approach for the gene therapy of Duchenne muscular dystrophy (DMD) is exon skipping. When thinking of possible intervention on human, it is very crucial to identify the most appropriate antisense sequences able to provide the highest possible skipping efficiency. In this article, we compared the exon 51 skipping activity of 10 different antisense molecules, raised against splice junctions and/or exonic splicing enhancers (ESEs), expressed as part of the U1 small nuclear RNA (snRNA). The effectiveness of each construct was tested in human DMD myoblasts carrying the deletion of exons 48-50, which can be treated with skipping of exon 51. Our results show that the highest skipping activity and dystrophin rescue is achieved upon expression of a U1 snRNA-derived antisense molecule targeting exon 51 splice sites in combination with an internal exon sequence. The efficacy of this molecule was further proven on an exon 45-50 deletion background, utilizing patient's fibroblasts transdifferentiated into myoblasts. In this system, we showed that the selected antisense was able to produce 50% skipping of exon 51.