Duchenne muscular dystrophy (DMD) is an X-linked inherited neuromuscular disorder due to mutations in the dystrophin gene. It is characterized by progressive muscle weakness and wasting due to the absence of dystrophin protein that causes degeneration of skeletal and cardiac muscle. The molecular diagnostic of DMD involves a deletions/duplications analysis performed by quantitative technique such as microarray-based comparative genomic hybridization (array-CGH), Multiple Ligation Probe Assay MLPA. Since traditional methods for detection of point mutations and other sequence variants require high cost and are time consuming, especially for a large gene like dystrophin, the use of next-generation sequencing (NGS) has become a useful tool available for clinical diagnosis. The dystrophin gene is large and finely regulated in terms of tissue expression, and RNA processing and editing includes a variety of fine tuned processes. At present, there are no effective treatments and the steroids are the only fully approved drugs used in DMD therapy able to slow disease progression. In the last years, an increasing variety of strategies have been studied as a possible therapeutic approach aimed to restore dystrophin production and to preserve muscle mass, ameliorating the DMD phenotype. RNA is the most studied target for the development of clinical strategies and Antisense Oligonucleotides (AONs) are the most used molecules for RNA modulation. The identification of delivery system to enhance the efficacy and to reduce the toxicity of AON is the main purpose in this area and nanomaterials are a very promising model as DNA/RNA molecules vectors. Dystrophinopathies therefore represent a pivotal field of investigation, which has opened novel avenues in molecular biology, medical genetics and novel therapeutic options. OPEN ACCESS

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity can result from alternate translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. This novel isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. IRES activity is confirmed in patient muscle by both peptide sequencing and ribosome profiling. Generation of a truncated reading frame upstream of the IRES by exon skipping leads to synthesis of a functional N-truncated isoform in both patient-derived cell lines and in a new DMD mouse model, where expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. These results support a novel therapeutic approach for patients with mutations within the 5’ exons of DMD.

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Background: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/ mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene.

in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein d o m a i n , w i t h e ff e c t s on p o p u l a t i o n ge n e t i c c h a r a c t e r i s t i c s a n d ne w personalized therapies.