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Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches.

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2′-O-methyl-phosphorothioate (2′OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.