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Summary Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD., We defined urinary stem cell (USC) RNA-seq signatures and provided evidence that they are a non-invasive, affordable cell source to study Duchenne muscular dystrophy (DMD), an X-linked disease, due to dystrophin gene mutations. Based on USCs’ RNA profiling and whole-genome sequencing (WGS), we identified three undetected atypical DMD mutations.

Absence of integral sarcolemmal protein, dystrophin, disrupts the RhoA-actin-SRF cascade in skeletal muscle, with subsequent dysregulation of downstream-SRF circadian targets and circadian rhythm., Absence of dystrophin, an essential sarcolemmal protein required for muscle contraction, leads to the devastating muscle-wasting disease Duchenne muscular dystrophy. Dystrophin has an actin-binding domain, which binds and stabilises filamentous-(F)-actin, an integral component of the RhoA-actin-serum-response-factor-(SRF) pathway. This pathway plays a crucial role in circadian signalling, whereby the suprachiasmatic nucleus (SCN) transmits cues to peripheral tissues, activating SRF and transcription of clock-target genes. Given dystrophin binds F-actin and disturbed SRF-signalling disrupts clock entrainment, we hypothesised dystrophin loss causes circadian deficits. We show for the first time alterations in the RhoA-actin-SRF-signalling pathway, in dystrophin-deficient myotubes and dystrophic mouse models. Specifically, we demonstrate reduced F/G-actin ratios, altered MRTF levels, dysregulated core-clock and downstream target-genes, and down-regulation of key circadian genes in muscle biopsies from Duchenne patients harbouring an array of mutations. Furthermore, we show dystrophin is absent in the SCN of dystrophic mice which display disrupted circadian locomotor behaviour, indicative of disrupted SCN signalling. Therefore, dystrophin is an important component of the RhoA-actin-SRF pathway and novel mediator of circadian signalling in peripheral tissues, loss of which leads to circadian dysregulation.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients’ plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

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.

Background: Neuromuscular disorders (NMDs) are a heterogeneous group of genetic diseases, caused by mutations in genes involved in spinal cord, peripheral nerve, neuromuscular junction, and muscle functions. To advance the knowledge of the pathological mechanisms underlying NMDs and to eventually identify new potential drugs paving the way for personalized medicine, limitations regarding the availability of neuromuscular disease-related biological samples, rarely accessible from patients, are a major challenge. Aim: We characterized urinary stem cells (USCs) by in-depth transcriptome and protein profiling to evaluate whether this easily accessible source of patient-derived cells is suitable to study neuromuscular genetic diseases, focusing especially on those currently involved in clinical trials. Methods: The global transcriptomics of either native or MyoD transformed USCs obtained from control individuals was performed by RNA-seq. The expression of 610 genes belonging to 16 groups of disorders (http://www.musclegenetable.fr/) whose mutations cause neuromuscular diseases, was investigated on the RNA-seq output. In addition, protein expression of 11 genes related to NMDs including COL6A, EMD, LMNA, SMN, UBA1, DYNC1H1, SOD1, C9orf72, DYSF, DAG1, and HTT was analyzed in native USCs by immunofluorescence and/or Western blot (WB). Results: RNA-seq profile of control USCs shows that 571 out of 610 genes known to be involved in NMDs, are expressed in USCs. Interestingly, the expression levels of the majority of NMD genes remain unmodified following USCs MyoD transformation. Most genes involved in the pathogenesis of all 16 groups of NMDs are well represented except for channelopathies and malignant hyperthermia related genes. All tested proteins showed high expression values, suggesting consistency between transcription and protein representation in USCs. Conclusion: Our data suggest that USCs are human cells, obtainable by non-invasive means, which might be used as a patient-specific cell model to study neuromuscular disease-causing genes and that they can be likely adopted for a variety of in vitro functional studies such as mutation characterization, pathway identification, and drug screening.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients’ plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model. In vitro cell-free analysis demonstrated a good loading capacity of PMO into NBs, while experiments in cell cultures showed lack of therapeutic effect since expression of DUX4 and its targets remained unmodified. We conclude that these types of chitosan-shelled NBs do not release PMO-AON and are therefore not ideal for PMO AON-related therapies.

Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model.

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 the TNFRSF10A gene. 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. The EU BIO-NMD project no. 241665 and the SOLVE-RD project (H2020 ID: 779257) were supported this study. FM was supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London. The support of the Muscular Dystrophy UK to the Dubowitz Neuromuscular Centre and of the MRC Neuromuscular Centre and BRC Biobank is also gratefully acknowledged. Part of this study was also supported by the NIHR grant “Rare Diseases Translational Research Collaboration” on Duchenne muscular dystrophy to FM. HL receives support from the Canadian Institutes of Health Research (Foundation Grant FDN-167281), the Canadian Institutes of Health Research and Muscular Dystrophy Canada (Network Catalyst Grant for NMD4C), the Canada Foundation for Innovation (CFI-JELF 38412), and the Canada Research Chairs program (Canada Research Chair in Neuromuscular Genomics and Health, 950-232279). The EUROBIOBANK, European Network of DNA, Cell and Tissue Banks for Rare Diseases was also acknowledged.

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008–2017). In DMD patients, we found deletions 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 domain, with effects on population genetic characteristics and new personalized therapies. © Copyright © 2020 Neri, Rossi, Trabanelli, Mauro, Selvatici, Falzarano, Spedicato, Margutti, Rimessi, Fortunato, Fabris, Gualandi, Comi, Tedeschi, Seia, Fiorillo, Traverso, Bruno, Giardina, Piemontese, Merla, Cau, Marica, Scuderi, Borgione, Tessa, Astrea, Santorelli, Merlini, Mora, Bernasconi, Gibertini, Sansone, Mongini, Berardinelli, Pini, Liguori, Filosto, Messina, Vita, Toscano, Vita, Pane, Servidei, Pegoraro, Bello, Travaglini, Bertini, D'Amico, Ergoli, Politano, Torella, Nigro, Mercuri and Ferlini

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.

Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting ~1:5000 live male births. Following the identification of pathogenic variations in the dystrophin gene in 1986, the underlining genotype/phenotype correlations emerged and the role of the dystrophin protein was elucidated in skeletal, smooth, and cardiac muscles, as well as in the brain. When the dystrophin protein is absent or quantitatively or qualitatively modified, the muscle cannot sustain the stress of repeated contractions. Dystrophin acts as a bridging and anchoring protein between the sarcomere and the sarcolemma, and its absence or reduction leads to severe muscle damage that eventually cannot be repaired, with its ultimate substitution by connective tissue and fat. The advances of an understanding of the molecular pathways affected in DMD have led to the development of many therapeutic strategies that tackle different aspects of disease etiopathogenesis, which have recently led to the first successful approved orphan drugs for this condition. The therapeutic advances in this field have progressed exponentially, with second-generation drugs now entering in clinical trials as gene therapy, potentially providing a further effective approach to the condition.

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.

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.

Background: Genetic alterations cause Hereditary Diseases (HDs) with a wide range of incidences. Some, like cystic fibrosis, occur frequently (1/1,000 newborns), whilst others, such as Pompe disease and other metabolic disorders are very rare (1/100,000 newborns). They are well under the threshold of 1/3,000, denoted by the European Community as Rare Diseases (RDs). Genetic alterations are also associated with multifactorial disorders like diabetes, and underline both somatic and germline mutations in cancer. Nowadays, thanks to the interventions of the European Union and the American National Health Institute as well as others, Hds are under an international lense, which has stimulated discussions and research targeting gene identification, prenatal diagnosis and care optimization leading to the development of new treatment options. Nanomedicine is paving the way toward some highly appealing clinical and research avenues in HDs. Nanotechnologies lend themselves to many aspects in human healthcare, such as in vitro diagnostics (nanobiosensors and nanoplatforms), drug delivery (nanovectors), drug monitoring (nanosensors) and artificial organs to study the genome variant meaning (nanostructures). Methods: and Results: With a significant reduction in costs and simplified healthcare delivery, nanodiagnostics can potentially provide the tools to diagnose diseases at an early stage with precision. In vitro nanodiagnostics are already diagnosing RDs, with many nanodevices having been successfully introduced over the last few decades. Nanovectors represent an emerging approach in drug delivery and treatment for several diseases such as cancers, infectious diseases, cardiovascular disorders and neurological pathologies. Artificial tissues have valuable implications in replacing compromised organs, thus offering unique opportunities to explore pathogenic mechanisms as well as new drug targets in a personalized context. Conclusion: This article outlines and discusses the recent progress in nanotechnology and its potential applications in HDs. It is a pivotal field for research and innovation in healthcare, with emphasis on diagnostics, disease monitoring, biomarker assaying and drug delivery. We underlined the nanomethod’s capacity to identify genetic alterations and the follow up of important aspects of the disease course, including therapies. We extensively described the new field of nanodelivery for experimental drugs, focusing on new genetic therapies and their implications in hereditary disorders. We also detailed innovative tools as artificial tissues based on nanomatrices and their use to identify or study genetic alterations.

Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy.

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb 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 16 h, 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 2.2 Mb 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.

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. Hum Mutat 33:572–581, 2012. © 2011 Wiley Periodicals, Inc.

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization.

Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.

We have previously demonstrated that intraperitoneal injections of 2′-O-methyl-phosphorothioate (2′OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles ofmdxmice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were trackedin vivoas well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, inmdxmice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

RNA is an important target for therapeutic approach of several diseases and the development of strategies to modulate its function has become the goal of many scientists. Antisense oligonucleotides (AONs) are the most used molecules for RNA targeting.

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.

We have tested different types of polymeric cationic core–shell nanoparticles (NPs) for delivering 2- O -methyl-phosphorothioate antisense oligoribonucleotides (AONs), in mdx mice. Both T1 and ZM2 NP bind and convey AONs: intraperitoneal (IP) injections of low doses (52.5 mg/kg) of NP–AON complex restored dystrophin protein synthesis in skeletal and cardiac muscles, allowing protein localization in up to 40% of muscle fibers with skipping level up to 20%. We have tested in vivo (mdx) the tissue biodistribution and elimination timing of NPs by Odyssey, using an infrared dye conjugated ZM2 NP. Administration mode was both IP and oral. Elimination by feces was up to 80% after 22 days of a single injection treatment in mdx treated by intraperitoneal doses, and close to 100% in those treated orally after 72 h of a single dose treatment. Evaluation of NP biodistribution, measured by Odyssey, in organs/tissues cryosections of sacrificed mdx revealed an intense positivity of labeled NP in muscles, heart, intestine, and all organs. We also demonstrated that NP–AON formulations passes the gastric barrier and induce dystrophin rescue in the intestinal smooth muscles as well as in the diaphragm. However the intense positivity at biodistribution of NPs in the heart (and skeletal muscles) conflicts with the relatively low efficacy of the compound in terms of skipping and dystrophin rescue. We are therefore performing ELISA assay to dose the antisense in the treated mice muscles in order to define the release kinetic of the AON from NPs. This is crucial in order to further proceed with other NP studies.

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.

In this paper we report the synthesis and the chemotaxis inhibitory activity of a number of 1H-pyrazole-4-carboxylic acid ethyl esters 2 functionalized in N1 with a methyl group or different hydroxyalkyl chains and in position 5 with a series of 3-substituted urea groups. These compounds were designed as development of previous pyrazole-urea derivatives that resulted potent IL8-induced neutrophil chemotaxis inhibitors in vitro. Most of the new compounds revealed a potent inhibition of both IL8- and fMLP-OMe-stimulated neutrophil chemotaxis. The most active compounds in the fMLP-OMe induced chemotaxis test showed IC(50) in the range 0.19 nM-2 microM; but we observed a very strong inhibition in the IL8-induced chemotaxis test, having the most active compounds IC(50) at pM concentrations. In vivo compounds 2e and 2f, although to a lesser extent, at 50mg/kg os decreased granulocyte infiltration in zymosan-induced peritonitis in mice.

It is well known that both acute and chronic autoimmune inflammatory disorders arise following a breakdown in control of neutrophil activation and recruitment. In the search for new anti-inflammatory agents, we synthesized some new 2-phenyl-2,3-dihydro-1H-imidazo[1,2-b]pyrazole derivatives and tested them in vitro in order to evaluate their ability to interfere with human neutrophil functions. All tested compounds showed strong inhibition of fMLP-OMe-induced chemotaxis, although they appeared unable to block degranulation and the fMLP-OMe-induced respiratory burst, and were inactive in binding experiments.

The two diastereomeric tripeptides f-(S)-HmMet-Leu-Phe-OMe and f-(R)-HmMet-Leu-Phe-OMe, analogues of the prototypical chemoattractant f-Met-Leu-Phe-OH, were synthesized in solution by classical methods and fully characterized. A conformational study was performed in solution by 1H-NMR. Concomitantly, the two peptides were tested for their ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The conformational and biological data are discussed with regard to the proposed model of the chemotactic receptor on neutrophils.

The two diastereomeric tripeptides f-(S)-HmMet-Leu-Phe-OMe and f-(R)-HmMet-Leu-Phe-OMe, analogues of the prototypical chemoattractant f-Met-Leu-Phe-OH, were synthesized in solution by classical methods and fully characterized. A conformational study was performed in solution by 1H-NMR. Concomitantly, the two peptides were tested for their ability to induce chemotaxis, superoxide anion production and lysozyme secretion from human neutrophils. The conformational and biological data are discussed with regard to the proposed model of the chemotactic receptor on neutrophils. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.

Frame-truncating mutations within the first five exons of the DMD gene typically do not result in Duchenne muscular dystrophy, but instead result in milder dystrophinopathy syndromes. This was first demonstrated in individuals carrying the c.9G > A (p.Trp3X) mutation, a North American founder allele associated with clinical severity ranging from a very mild Becker muscular dystrophy phenotype to a complete absence of symptoms. We have previously shown that the mild phenotype associated with this and similar 5’ exon mutations is due to alternative initiation of translation from methionines encoded in exon 6, a process mediated by the presence of a muscle-specific and glucocorticoid-inducible internal ribosomal entry site (IRES) found within exon 5. The resultant N-truncated dystrophin protein lacks the first calponin homology domain of the canonical actin binding domain 1. Nevertheless, it is highly functional, raising the possibility of the therapeutic use of this isoform. We hypothesized that disruption of the mRNA open reading frame via exon skipping – resulting in a downstream premature termination codon – would stimulate IRES utilization. The feasibility of this approach is supported by the recent identification of an asymptomatic patient harbouring an exon 2 deletion, and we designed U7snRNA vectors targeting exon 2 for use in both patient-derived cell lines and in a new DMD mouse model harbouring an exon 2 duplication. Both in vitro and in vivo we can stimulate IRES activity, and by combining exon-skipping and glucocorticoid treatment we are able to restore expression of a properly localized yet N-truncated dystrophin. These results not only provide evidence for the functionality of the dystrophin IRES – and, hence, for the presence of a novel N-truncated dystrophin isoform under yet to be clarified physiologic conditions – but also suggest that out-of-frame skipping is a promising therapeutic approach for DMD patients harbouring 5’ mutations.

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.