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Aims A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. Methods and results Echocardiography showed normal cardiac structure and function in 12–13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. The absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1–2 by quantitative polymerase chain reaction) and an increase in NOS1 polyubiquitination [assessed using tandem polyubiquitination pulldowns; P < 0.05 vs. wild type (WT)]. Neither the up-regulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their WT littermates. Conclusion Dystrophin depletion is not associated with atrial miR-31 up-regulation, reduced NOS activity, or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content. [ABSTRACT FROM AUTHOR]

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2′-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k- mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a currently incurable X-linked neuromuscular disorder, characterized by progressive muscle wasting and premature death, typically as a consequence of cardiac failure. DMD-causing mutations in the dystrophin gene are highly diverse, meaning that the development of a universally-applicable therapy to treat all patients is very challenging. The leading therapeutic strategy for DMD is antisense oligonucleotide-mediated splice modulation, whereby one or more specific exons are excluded from the mature dystrophin mRNA in order to correct the translation reading frame. Indeed, three exon skipping oligonucleotides have received FDA approval for use in DMD patients. Second-generation exon skipping drugs (i.e. peptide-antisense oligonucleotide conjugates) exhibit enhanced potency, and also induce dystrophin restoration in the heart. Similarly, multiple additional antisense oligonucleotide drugs targeting various exons are in clinical development in order to treat a greater proportion of DMD patient mutations. Relatively recent advances in the field of genome engineering (specifically, the development of the CRISPR/Cas system) have provided multiple promising therapeutic approaches for the RNA-directed genetic correction of DMD, including exon excision, exon reframing via the introduction of insertion/deletion mutations, disruption of splice signals to promote exon skipping, and the templated correction of point mutations by seamless homology directed repair or base editing technology. Potential limitations to the clinical translation of the splice modulation and gene editing approaches are discussed, including drug delivery, the importance of uniform dystrophin expression in corrected myofibres, safety issues (e.g. renal toxicity, viral vector immunogenicity, and off-target gene editing), and the high cost of therapy. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy (DMD) is a fatal muscle‐wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex‐miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex‐miRNAs (e.g. miR‐1, miR‐133a, miR‐206, and miR‐483) are highly up‐regulated in the serum of DMD patients and dystrophic animal models and are restored to wild‐type levels following exon skipping‐mediated dystrophin rescue in mdx mice. As such, ex‐miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex‐miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. Methods: Candidate ex‐miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide‐PMO (PPMO) exon skipping conjugates and in mdx‐XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X‐chromosome inactivation. miRNA profiling was performed in mdx‐XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT‐qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. Results: The restoration of ex‐myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin‐expressing mdx‐XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild‐type levels). Instead, ex‐miRNAs were present at high levels in mdx‐XistΔhs mice regardless of dystrophin expression. PPMO‐treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx‐XistΔhs muscles showed non‐homogeneous dystrophin staining and sporadic regenerating foci. Conclusions: Uniform dystrophin expression is required to prevent ex‐miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle. [ABSTRACT FROM AUTHOR]

Cardiac failure is a major cause of mortality in patients with Duchenne muscular dystrophy (DMD). Antisense-mediated exon skipping has the ability to correct out-of-frame mutations in DMD to produce truncated but functional dystrophin. Traditional antisense approaches have however been limited by their poor uptake into cardiac muscle. The addition of cell-penetrating peptides to antisense molecules has increased their potency and improved their uptake into all muscles, including the heart. We have investigated the efficacy of the Peptide-conjugated phosphodiamidate morpholino oligomer (P-PMO) Pip6a-PMO, for restoration of cardiac dystrophin and functional rescue in DMD mice- the mdx mouse and the less well characterised Cmah-/-mdx mouse (which carry a human-like mutation in the mouse Cmah gene as well as a mutation in DMD). In our first study male mdx mice were administered Pip6a-PMO, i.v, fortnightly from 12 to 30 weeks of age alongside mock-injected age-matched mdx and C57BL10 controls. Mice received 4 doses of 18 mg/kg followed by 8 doses of 12.5 mg/kg. The cardiac function of the mice was analysed 2 weeks after their final injection by MRI followed by conductance catheter and their muscles were harvested for dystrophin quantification. In the second study, male Cmah-/-mdx mice, received 12.5 mg/kg Pip6a-PMO, i.v fortnightly from 8 to 26 weeks and assessed by MRI at 3 time points (12, 18 and 28 weeks) alongside mock-injected age-matched mdx, C57BL10 and Cmah-/-mdx controls. The mice also underwent MEMRI and conductance catheter at 28 weeks. This allowed us to characterise the cardiac phenotype of Cmah-/-mdx mice as well as assess the effects of P-PMO on cardiac function. Pip6a-PMO treatment resulted in significant restoration of dystrophin in mdx and Cmah-/-mdx mice (37.5% and 51.6%, respectively), which was sufficient to significantly improve cardiac function, ameliorating both right and left ventricular dysfunction. Cmah-/-mdx mice showed an abnormal response to dobutamine stress test and this was completely ameliorated by PIP6a-PMO treatment. These encouraging data suggest that total restoration of dystrophin may not be required to significantly improve cardiac outcome in DMD patients and that it may be realistic to expect functional improvements with modest levels of dystrophin restoration which may be very achievable in future clinical trials. [ABSTRACT FROM AUTHOR]

miRNAs are small, noncoding RNAs that not only regulate gene expression within cells, but might also constitute promising extracellular biomarkers for a variety of pathologies, including the progressive muscle-wasting disorder Duchenne Muscular Dystrophy (DMD). A set of muscle-enriched miRNAs, the myomiRs (miR-1, miR-133, and miR-206) are highly elevated in the serum of patients with DMD and in dystrophin-deficient animal models. Furthermore, circulating myomiRs might be used as pharmacodynamic biomarkers, given that their levels can be restored towards wild-type levels following exon skipping therapy in dystrophic mice. The relationship between muscle pathology and extracellular myomiR release is complex, and incompletely understood. Here, we discuss current progress leading towards the clinical utility of extracellular miRNAs as putative DMD biomarkers, and their possible contribution to muscle physiology. [ABSTRACT FROM AUTHOR]

Extracellular microRNAs (miRNAs) are under investigation as minimally-invasive biomarkers for a wide range of disease conditions. We have recently shown in a mouse model of the progressive muscle-wasting condition Duchenne muscular dystrophy (DMD) that a set of highly elevated serum miRNAs reflects the regenerative status of muscle. These miRNAs are promising biomarkers for monitoring DMD disease progression and the response to experimental therapies. The gold standard miRNA detection methodology is Reverse Transcriptase-quantitative Polymerase Chain Reaction (RT-qPCR), which typically exhibits high sensitivity and wide dynamic range. Accurate determination of miRNA levels is affected by RT-qPCR normalization method and therefore selection of the optimal strategy is of critical importance. Serum miRNA abundance was measured by RT-qPCR array in 14 week old mice, and by individual RT-qPCR assays in a time course experiment spanning 48 weeks. Here we utilize these two datasets to assess the validity of three miRNA normalization strategies (a) normalization to the average of all Cq values from array experiments, (b) normalization to a stably expressed endogenous reference miRNA, and (c) normalization to an external spike-in synthetic oligonucleotide. Normalization approaches based on endogenous control miRNAs result in an under-estimation of miRNA levels by a factor of ∼2. An increase in total RNA and total miRNA was observed in dystrophic serum which may account for this systematic bias. We conclude that the optimal strategy for this model system is to normalize to a synthetic spike-in control oligonucleotide. [ABSTRACT FROM AUTHOR]

Abstract: Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. [Copyright &y& Elsevier]

Duchenne muscular dystrophy (DMD) is a devastating progressive disease for which there is currently no effective treatment except palliative therapy. There are several promising genetic approaches, including viral delivery of the missing dystrophin gene, read-through of translation stop codons, exon skipping to restore the reading frame and increased expression of the compensatory utrophin gene. The lessons learned from these approaches will be applicable to many other disorders. [ABSTRACT FROM AUTHOR]

AbstractDuchenne muscular dystrophy (DMD) is a severe muscle wasting disorder caused by mutations in the DMDgene, affecting 1 in 3500 newborn males. Complete loss of muscle dystrophin protein causes progressive muscle weakness and heart and respiratory failure, leading to premature death. Antisense oligonucleotides (AONs) that bind to complementary sequences of the dystrophin pre-mRNA to induce skipping of the targeted exon by modulating pre-mRNA splicing are promising therapeutic agents for DMD. Such AONs can restore the open reading frame of the DMDgene and produce internally deleted, yet partially functional dystrophin protein isoforms in skeletal muscle. Within the last few years, clinical trials using AONs have made considerable progress demonstrating the restoration of functional dystrophin protein and acceptable safety profiles following both local and systemic delivery in DMD patients. However, improvement of AON delivery and efficacy, along with the development of multiple AONs to treat as many DMD patients as possible needs to be addressed for this approach to fulfill its potential. Here, we review the recent progress made in clinical trials using AONs to treat DMD and discuss the current challenges to the development of AON-based therapy for DMD. [ABSTRACT FROM AUTHOR]

The development of effective therapies for neuromuscular disorders such as Duchenne muscular dystrophy (DMD) is hampered by considerable challenges: skeletal muscle is the most abundant tissue in the body, and many neuromuscular disorders are multisystemic conditions. However, despite these barriers there has recently been substantial progress in the search for novel treatments. In particular, the use of antisense oligonucleotides, which are designed to target RNA and modulate pre-mRNA splicing to restore functional protein isoforms or directly inhibit the toxic effects of pathogenic RNAs, offers great promise and these approaches are now being tested in the clinic. Here, we review recent advances in the development of such antisense oligonucleotides and other promising novel approaches, including the induction of readthrough nonsense mutations. [ABSTRACT FROM AUTHOR]

Splice-modulation therapy, whereby molecular manipulation of premessenger RNA splicing is engineered to yield genetic correction, is a promising novel therapy for genetic diseases of muscle and nerve—the prototypical example being Duchenne muscular dystrophy. Duchenne muscular dystrophy is the most common childhood genetic disease, affecting one in 3500 newborn boys, causing progressive muscle weakness, heart and respiratory failure and premature death. No cure exists for this disease and a number of promising new molecular therapies are being intensively studied. Duchenne muscular dystrophy arises due to mutations that disrupt the open-reading-frame in the DMD gene leading to the absence of the essential muscle protein dystrophin. Of all novel molecular interventions currently being investigated for Duchenne muscular dystrophy, perhaps the most promising method aiming to restore dystrophin expression to diseased cells is known as ‘exon skipping’ or splice-modulation, whereby antisense oligonucleotides eliminate the deleterious effects of DMD mutations by modulating dystrophin pre-messenger RNA splicing, such that functional dystrophin protein is produced. Recently this method was shown to be promising and safe in clinical trials both in The Netherlands and the UK. These trials studied direct antisense oligonucleotide injections into single peripheral lower limb muscles, whereas a viable therapy will need antisense oligonucleotides to be delivered systemically to all muscles, most critically to the heart, and ultimately to all other affected tissues including brain. There has also been considerable progress in understanding how such splice-correction methods could be applied to the treatment of related neuromuscular diseases, including spinal muscular atrophy and myotonic dystrophy, where defects of splicing or alternative splicing are closely related to the disease mechanism. [ABSTRACT FROM PUBLISHER]

Duchenne muscular dystrophy (DMD) is caused by absence of the integral structural protein, dystrophin, which renders muscle fibres susceptible to injury and degeneration. This ultimately results in cardiorespiratory dysfunction, which is the predominant cause of death in DMD patients, and highlights the importance of therapeutic targeting of the cardiorespiratory system. While there is some evidence to suggest that restoring dystrophin in the diaphragm improves both respiratory and cardiac function, the role of the diaphragm is not well understood. Here using exon skipping oligonucleotides we predominantly restored dystrophin in the diaphragm and assessed cardiac function by MRI. This approach reduced diaphragmatic pathophysiology and markedly improved diaphragm function but did not improve cardiac function or pathophysiology, with or without exercise. Interestingly, exercise resulted in a reduction of dystrophin protein and exon skipping in the diaphragm. This suggests that treatment regimens may require modification in more active patients. In conclusion, whilst the diaphragm is an important respiratory muscle, it is likely that dystrophin needs to be restored in other tissues, including multiple accessory respiratory muscles, and of course the heart itself for appropriate therapeutic outcomes. This supports the requirement of a body-wide therapy to treat DMD. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is a rare, progressive, muscle-wasting disease leading to severe disability and premature death. Treatment is currently symptomatic, but several experimental therapies are in development. Implemented care standards, validated outcome measures correlating with clinical benefit, and comprehensive information about the natural history of the disease are essential for regulatory approval of any treatment. However, for Duchenne muscular dystrophy and other rare diseases, these requirements are not always in place when potential therapies enter the clinical trial phase. A cooperative effort of stakeholders in Duchenne muscular dystrophy-including representatives from patients' groups, academia, industry, and regulatory agencies-is aimed at addressing this shortfall by identifying strategies to overcome challenges, developing the tools needed, and collecting relevant data. An open and constructive dialogue among European stakeholders has positively affected development of treatments for Duchenne muscular dystrophy; this approach could serve as a paradigm for development of treatments for rare diseases in general. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (ASOs) have the potentialto revolutionize medicine due to their ability to manipulate genefunction for therapeutic purposes. ASOs are chemically modified and/orincorporated within nanoparticles to enhance their stability and cellularuptake, however, a major challenge is the poor understanding of theiruptake mechanisms, which would facilitate improved ASO designs withenhanced activity and reduced toxicity. Here, we study the uptakemechanism of three therapeutically relevant ASOs (peptide-conjugatedphosphorodiamidate morpholino (PPMO), 2′Omethyl phosphorothioate(2′OMe), and phosphorothioated tricyclo DNA (tcDNA) that havebeen optimized to induce exon skipping in models of Duchenne musculardystrophy (DMD). We show that PPMO and tcDNA have high propensityto spontaneously self-assemble into nanoparticles. PPMO forms micellesof defined size and their net charge (zeta potential) is dependenton the medium and concentration. In biomimetic conditions and at lowconcentrations, PPMO obtains net negative charge and its uptake ismediated by class A scavenger receptor subtypes (SCARAs) as shownby competitive inhibition and RNAi silencing experiments in vitro.In vivo, the activity of PPMO was significantly decreased in SCARA1knockout mice compared to wild-type animals. Additionally, we showthat SCARA1 is involved in the uptake of tcDNA and 2′OMe asshown by competitive inhibition and colocalization experiments. Surfaceplasmon resonance binding analysis to SCARA1 demonstrated that PPMOand tcDNA have higher binding profiles to the receptor compared to2′OMe. These results demonstrate receptor-mediated uptake fora range of therapeutic ASO chemistries, a mechanism that is dependenton their self-assembly into nanoparticles. [ABSTRACT FROM AUTHOR]

The article presents a dose-escalation study that investigates the effect of phosphorodiamidate morpholino oligomer treatment in patients with Duchenne muscular dystrophy. It states that the method of the study includes a muscle biopsy of the patients before starting with the treatment and after 12 weekly intravenous infusions of AVI-4658. It reveals the safety and biochemical efficacy of AVI-4658 as a disease modifying drug for Duchenne muscular dystrophy.

Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics. [ABSTRACT FROM AUTHOR]

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy. [ABSTRACT FROM AUTHOR]

Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. [ABSTRACT FROM AUTHOR]

Summary: Background: Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods: We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings: Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation: Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding: UK Department of Health. [Copyright &y& Elsevier]

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two main mechanisms, the classical and trans-signalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 trans-signalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs. [ABSTRACT FROM AUTHOR]