Rapid, comprehensive analysis of the dystrophin transcript by a custom micro-fluidic exome array

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mRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients' cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients' myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies

Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2 -O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules

Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2  -O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules

Exon Skipping-Mediated Dystrophin Reading Frame Restoration for Small Mutations

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Tumor Necrosis Factor Receptor SF10A (TNFRSF10A) SNPs Correlate With Corticosteroid Response in Duchenne Muscular Dystrophy

Background Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. Methods and Findings We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in theTNFRSF10Agene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N= 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. Conclusion We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker

A multicenter comparison of quantification methods for antisense oligonucleotide-induced DMD exon 51 skipping in Duchenne muscular dystrophy cell cultures

Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR

Dystrophin regulates peripheral circadian SRF signalling

Dystrophin is a sarcolemmal protein essential for muscle contraction and maintenance, absence of which leads to the devastating muscle wasting disease Duchenne muscular dystrophy (DMD)[1, 2]. Dystrophin has an actin-binding domain [3–5], which specifically binds and stabilises filamentous (F)-actin[6], an integral component of the RhoA-actin-serum response factor (SRF)-pathway[7]. The RhoA-actin-SRF-pathway plays an essential role in circadian signalling whereby the hypothalamic suprachiasmatic nucleus, transmits systemic cues to peripheral tissues, activating SRF and transcription of clock target genes[8, 9]. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised that dystrophin loss causes circadian deficits. Here we show for the first time alterations in the RhoA-actin-SRF-signalling-pathway, in both dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios and nuclear MRTF, dysregulation of core clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from DMD patients harbouring an array of mutations. Further, disrupted circadian locomotor behaviour was observed in dystrophic mice indicative of disrupted SCN signalling, and indeed dystrophin protein was absent in the SCN of dystrophic animals. Dystrophin is thus a critically important component of the RhoA-actin-SRF-pathway and a novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation

Urinary Stem Cells as Tools to Study Genetic Disease: Overview of the Literature

Urine specimens represent a novel and non-invasive approach to isolate patient-specific stem cells by easy and low-cost procedures, replacing the traditional sources (muscle/skin biopsy/adipose tissue) obtained with invasive and time-consuming methods. Urine-derived stem cells (USCs) can be used in a broad field of applications, such as regenerative medicine, cell therapy, diagnostic testing, disease modelling and drug screening. USCs are a good source of cells for generating induced pluripotent stem cells (iPSCs) and importantly, they can also be directly converted into specific cell lines. In this review, we show the features of USCs and their use as a promising in vitro model to study genetic diseases

Oligomeric formylpeptide activity on human neutrophils

A series of oligomeric formylpeptides were synthesized by cross-linking the prototype fMLP using a Lys residue. They were then investigated for their ability to stimulate chemotaxis, superoxide anion production, and lytic enzyme release in human neutrophils. Although active in stimulating the different receptor isoforms, leading to the different biological responses, these analogues showed a lesser potency and affinity than the standard peptide. On the basis of the results reported here, we can hypothesise that: (i) the increased bulk of these molecules seems to hinder their correct allocation into the receptor pocket, thereby hindering favourable receptor interaction; and that: (ii) fMLP space positions do not seem to allow the ligand to increase biological responses