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Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size., (© 2024. The Author(s).)

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies., (Copyright © 2017. Published by Elsevier B.V.)

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 has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

Background: Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial., Methods: Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females., Results: The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers., Conclusions: This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated independently. Moreover, neither the total DMD transcript level, nor the relative proportion of the wild-type transcript do correlate with the symptomatic phenotype.

Background: In X-linked dilated cardiomyopathy due to dystrophin mutations which abolish the expression of the M isoform (5'-XLDC), the skeletal muscle is spared through the up-regulation of the Brain (B) isoform, a compensatory mechanism that does not appear to occur in the heart of affected individuals., Methods: We quantitatively studied the expression topography of both B and M isoforms in various human heart regions through in-situ RNA hybridization, Reverse-Transcriptase and Real-Time PCR experiments. We also investigated the methylation profile of the B promoter region in the heart and quantified the B isoform up regulation in the skeletal muscle of two 5'-XLDC patients., Results: Unlike the M isoform, consistently detectable in all the heart regions, the B isoform was selectively expressed in atrial cardiomyocytes, but absent in ventricles and in conduction system structures. Although the level of B isoform messenger in the skeletal muscle of 5'-XLDC patients was lower that of the M messenger present in control muscle, it seems sufficient to avoid an overt muscle pathology. This result is consistent with the protein level in XLDC patients muscles we previously quantified. Methylation studies revealed that the B promoter shows an overall low level of methylation at the CG dinucleotides in both atria and ventricles, suggesting a methylation-independent regulation of the B promoter activity., Conclusions: The ventricular dilatation seen in 5'-XLDC patients appears to be functionally related to loss of the M isoform, the only isoform transcribed in human ventricles; in contrast, the B isoform is well expressed in heart but confined to the atria. Since the B isoform can functionally replace the M isoform in the skeletal muscle, its expression in the heart could potentially exert the same rescue function. Methylation status does not seem to play a role in the differential B promoter activity in atria and ventricles, which may be governed by other regulatory mechanisms. If these mechanisms could be deduced, de-silencing of the B isoform may represent a therapeutic strategy in 5'-XLDC patients., (© 2012 Neri et al; licensee BioMed Central Ltd.)

Duchenne and Becker muscular dystrophies are caused by mutations in the dystrophin gene. Both the enormous size of this gene and heterogeneous set of causative mutations behind these pathologies may hamper and even prevent accurate molecular diagnosis. Often RNA analysis is required not only to identify mutations escaping MLPA/CGH or exon sequencing but also to validate the functional effect of novel variations that may affect the exon composition of the DMD gene. We present the design and experimental validation of a new, simple, and easy-to-use platform we call FluiDMD. This platform is based on the Applied Biosystems 7900HT TaqMan(®) low-density array technology and is able to define the full-exon composition, profile the dystrophin isoforms present, establish changes in mRNA decay, and potentially identify all deletions/duplications and splicing affecting mutations contemporaneously. Moreover, we demonstrate that this system accurately detects the pathogenic effect of all dystrophin mutations belonging to any category, thereby highlighting the functional validation capacity of this system. The high efficacy and sensitivity of this tool in detecting mutations in the dystrophin transcript can be exploited in a variety of cells/tissues, in particular skin, which is harvested by causing minimum patient discomfort. We therefore propose FluiDMD as a validated diagnostic biomarker for molecular profiling of dystrophinopathies., (© 2011 Wiley Periodicals, Inc.)

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.

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.

Exon skipping using antisense oligonucleotides (AONs) has successfully been used to reframe the mRNA in various Duchenne muscular dystrophy patients carrying deletions in the DMD gene. In this study we tested the feasibility of the exon skipping approach for patients with small mutations in in-frame exons. We first identified 54 disease-causing point mutations. We selected five patients with nonsense or frameshifting mutations in exons 10, 16, 26, 33, and 34. Wild-type and mutation specific 2'OMePS AONs were tested in cell-free splicing assays and in cultured cells derived from the selected patients. The obtained results confirm cell-free splicing assay as an alternative system to test exon skipping propensity when patients' cells are unavailable. In myogenic cells, similar levels of exon skipping were observed for wild-type and mutation specific AONs for exons 16, 26, and 33, whereas for exon 10 and exon 34 the efficacy of the AONs was significantly different. Interestingly, in some cases skipping efficiencies for mutated exons were quite dissimilar when compared with previous reports on the respective wild-type exons. This behavior may be related to the effect of the mutations on exon skipping propensity, and highlights the complexity of identifying optimal AONs for skipping exons with small mutations.

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.

DMD gene exons duplications account for up to 5-10 % of Duchenne (DMD) and up to 5-19% of Becker (BMD) muscular dystrophies; as for the more common deletions, the genotype-phenotype correlation and the genetic prognosis are generally based on the "reading frame rule". Nevertheless, the transcriptional profile of duplications, abridging the genomic configuration to the eventual protein effect, has been poorly studied. We describe 26 DMD gene duplications occurring in 33 unrelated patients and detected among a cohort of 194 mutation-positive DMD/BMD patients. We have characterized at the RNA level 16 of them. Four duplications (15%) behave as exception to the reading frame rule. In three BMD cases with out-of-frame mutations, the RNA analysis revealed that exon skipping events occurring in the duplicated region represent the mechanism leading to the frame re-establishment and to the milder phenotype. Differently, in a DMD patient carrying an in-frame duplication the RNA behaviour failed to explain the clinical phenotype which is probably related to post-transcriptional-translational mechanisms. We conclude that defining the RNA profile in DMD gene duplications is mandatory both for establishing the genetic prognosis and for approaching therapeutic trials based on hnRNA modulation., ((c) 2008 Wiley-Liss, Inc.)

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., Results: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis., Conclusion: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.