Dystrophin In Duchenne Muscular Dystrophy Research Articles

The Spectrum of Deletion Pattern in the Dystrophin Gene in Duchenne Muscular Dystrophy Patients: A Cross-sectional Study from Northeast India

Introduction: Duchenne Muscular Dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive, irreversible muscle weakness. It is caused by a mutation in the dystrophin or DMD gene, leading to the absence of the essential muscle protein Dystrophin in DMD. Muscle strength continually diminishes, and death usually occurs from chronic respiratory insufficiency and/or cardiac failure. Due to the high mortality rate, early diagnosis is crucial to allow appropriate planning for patient care and treatment. In India, the traditional multiplex Polymerase Chain Reaction (PCR) assay is the most common method to detect Dystrophin gene deletion mutations. Aim: To describe the spectrum of deletion patterns in the dystrophin gene in muscular dystrophy patients attending a tertiary care hospital in Northeast India. Materials and Methods: A hospital-based cross-sectional study was conducted on a total of 53 suspected DMD patients attending the Department of Neurology and Department of Paediatrics at Assam Medical College, Dibrugarh, Assam, India from January 2016 to December 2022. Multiplex PCR was performed to study the deletion patterns for the 25 most common exons of the DMD gene for all patients at the Genetic lab, Department of Anatomy. Deletion mutations at different multiple exons were found, and the results were statistically analysed using Statistical Package for Social Sciences (SPSS) to calculate the mean and standard deviation. The results were presented in tabular form as percentages. Results: Out of the 53 cases suspected to have DMD on the basis of clinical presentation and high serum Creatine Kinase (CK) levels, 34 patients (64.2%) showing deletion mutations in the 25 most common exons of the DMD gene were included in the study. Deletions were most common in 15 (44.1%) patients in the distal hotspot region of exons 44-55. The most common gene deleted was exon 50 in 11 (32.4%) patients. The age at which symptoms were noticed was 4.7±2.01 years. The mean age at diagnosis was 8.4±2.4 years. Conclusion: In the present study, most patients suspected of DMD based on clinical and laboratory findings had deletions in the DMD gene, with the most common region for deletion in the dystrophin gene being the distal hotspot region, and exon 50 being the most commonly deleted.

Next steps for the optimization of exon skipping therapy for Duchenne muscular dystrophy

Introduction It is established that the exon skipping approach can restore dystrophin in Duchenne muscular dystrophy (DMD) patients. However, dystrophin restoration levels are low, and the field is evolving to provide solutions for improved exon skipping. DMD is a neuromuscular disorder associated with chronic muscle tissue loss attributed to the lack of dystrophin, which causes muscle inflammation, fibrosis formation, and impaired regeneration. Currently, four antisense oligonucleotides (AONs) based on phosphorodiamidate morpholino oligomer (PMO) chemistry are approved by US Food and Drug Administration for exon skipping therapy of eligible DMD patients. Areas covered This review describes a preclinical and clinical experience with approved and newly developed AONs for DMD, outlines efforts that have been done to enhance AON efficiency, reviews challenges of clinical trials, and summarizes the current state of the exon skipping approach in the DMD field. Expert opinion The exon skipping approach for DMD is under development, and several chemical modifications with improved properties are under (pre)-clinical investigation. Despite existing advantages of these modifications, their safety and effectiveness have to be examined in clinical trials, which are planned or ongoing. Furthermore, we propose clinical settings using natural history controls to facilitate studying the functional effect of the therapy.

Pharmacological inhibition of HDAC6 improves muscle phenotypes in dystrophin-deficient mice by downregulating TGF-β via Smad3 acetylation

The absence of dystrophin in Duchenne muscular dystrophy disrupts the dystrophin-associated glycoprotein complex resulting in skeletal muscle fiber fragility and atrophy, associated with fibrosis as well as microtubule and neuromuscular junction disorganization. The specific, non-conventional cytoplasmic histone deacetylase 6 (HDAC6) was recently shown to regulate acetylcholine receptor distribution and muscle atrophy. Here, we report that administration of the HDAC6 selective inhibitor tubastatin A to the Duchenne muscular dystrophy, mdx mouse model increases muscle strength, improves microtubule, neuromuscular junction, and dystrophin-associated glycoprotein complex organization, and reduces muscle atrophy and fibrosis. Interestingly, we found that the beneficial effects of HDAC6 inhibition involve the downregulation of transforming growth factor beta signaling. By increasing Smad3 acetylation in the cytoplasm, HDAC6 inhibition reduces Smad2/3 phosphorylation, nuclear translocation, and transcriptional activity. These findings provide in vivo evidence that Smad3 is a new target of HDAC6 and implicate HDAC6 as a potential therapeutic target in Duchenne muscular dystrophy.

VP.86 Effect of a chronic treatment with L-citrulline on funtional, histological and molecular readouts of dystrophic mdx mouse model

The absence of protein dystrophin in Duchenne muscular dystrophy leads to progressive muscle weakness and wasting, paralleled by failing regeneration. Recently, a key role of deregulated nitric oxide production in epigenetic control of regeneration program has been demonstrated. We focused on the potential interest of the amino acid L-citrulline (L-Cit), a precursor of L-arginine, physiologically required for protein turnover and nitric oxide production in muscle and blood vessels. We administered L-citrulline (2 mg/g/die), through diet, alone or in combination with PDN, (1 mg/kg, 5 days/week subcutaneously) to 4-5 week-old mdx mice for 8 weeks. We found that the increment of maximal forelimb strength was improved in all treated groups vs. untreated mdx mice, particularly by L-Cit alone or with PDN. Ex vivo, treated mdx mice displayed a significant increase of specific isometric twitch and tetanic force in diaphragm (DIA), with minor effects in extensor digitorum longus muscle. Additionally, L-Cit, alone or in combination with PDN, reduced fibrosis and inflammation in gastrocnemius and DIA muscles, and decreased plasma levels of lactate dehydrogenase. PCR analysis in muscles is currently ongoing. These data are encouraging regarding the potential benefits of L-Cit supplementation in DMD also in combination with standard therapy (NL-DPP grant).

VP.24 T Cell-mediated immune response to dystrophin in Duchenne muscular dystrophy - A natural history study

Duchenne muscular dystrophy is caused by a lack of dystrophin. Several strategies exist to restore dystrophin production, including exon skipping and gene therapy. However, dystrophin epitopes, expressed in revertant fibres in non-treated patients, may elicit a T cell-mediated immune response, which could reduce the efficacy of therapies designed to restore dystrophin. We performed enzyme-linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 78 paediatric patients with DMD from a natural history study. Immune responses to dystrophin were quantified using 368 peptides spanning the entire protein, organized into nine pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Overall, our results show that pre-existing T cell responses to dystrophin are uncommon (8%), inconsistent and low-level. Whilst this does provide some confidence for dystrophin restorative treatments, the fact that some patients are responsive warrants that individuals be screened prior to treatment.

The gRNA Vector Level Determines the Outcome of Systemic AAV CRISPR Therapy for Duchenne Muscular Dystrophy.

Adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR) editing holds promise to restore missing dystrophin in Duchenne muscular dystrophy (DMD). Intramuscular coinjection of CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) vectors resulted in robust dystrophin restoration in short-term studies in the mdx mouse model of DMD. Intriguingly, this strategy failed to yield efficient dystrophin rescue in muscle in a long-term (18-month) systemic injection study. In-depth analyses revealed a selective loss of the gRNA vector after long-term systemic, but not short-term local injection. To determine whether preferential gRNA vector depletion is due to the mode of delivery (local vs. systemic) or the duration of the study (short term vs. long term), we conducted a short-term systemic injection study. The gRNA (4e12 vg/mouse in the 1:1 group or 1.2e13 vg/mouse in the 3:1 group) and Cas9 (4e12 vg/mouse) vectors were coinjected intravenously into 4-week-old mdx mice. The ratio of the gRNA to Cas9 vector genome copy dropped from 1:1 and 3:1 at injection to 0.4:1 and 1:1 at harvest 3 months later, suggesting that the route of administration, rather than the experimental duration, determines preferential gRNA vector loss. Consistent with our long-term systemic injection study, the vector ratio did not influence Cas9 expression. However, the 3:1 group showed significantly higher dystrophin expression and genome editing, better myofiber size distribution, and a more pronounced improvement in muscle function and electrocardiography. Our data suggest that the gRNA vector dose determines the outcome of systemic AAV CRISPR therapy for DMD.

A Spotlight on T Lymphocytes in Duchenne Muscular Dystrophy-Not Just a Muscle Defect.

The lack of dystrophin in Duchenne muscular dystrophy (DMD) results in membrane fragility resulting in contraction-induced muscle damage and subsequent inflammation. The impact of inflammation is profound, resulting in fibrosis of skeletal muscle, the diaphragm and heart, which contributes to muscle weakness, reduced quality of life and premature death. To date, the innate immune system has been the major focus in individuals with DMD, and our understanding of the adaptive immune system, specifically T cells, is limited. Targeting the immune system has been the focus of multiple clinical trials for DMD and is considered a vital step in the development of better treatments. However, we must first have a complete picture of the involvement of the immune systems in dystrophic muscle disease to better understand how inflammation influences disease progression and severity. This review focuses on the role of T cells in DMD, highlighting the importance of looking beyond skeletal muscle when considering how the loss of dystrophin impacts disease progression. Finally, we propose that targeting T cells is a potential novel therapeutic in the treatment of DMD.

Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy.

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β-Glucans as Dietary Supplement to Improve Locomotion and Mitochondrial Respiration in a Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration. A lack of dystrophin in DMD leads to inflammatory response, autophagic dysregulation, and oxidative stress in skeletal muscle fibers that play a key role in the progression of the pathology. β-glucans can modulate immune function by modifying the phagocytic activity of immunocompetent cells, notably macrophages. Mitochondrial function is also involved in an important mechanism of the innate and adaptive immune responses, owing to high need for energy of immune cells. In the present study, the effects of 1,3-1,6 β-glucans on five-day-old non-dystrophic and dystrophic (sapje) zebrafish larvae were investigated. The effects of the sonication of β-glucans and the dechorionation of embryos were also evaluated. The results showed that the incidence of dystrophic phenotypes was reduced when dystrophic embryos were exposed to 2 and 4 mg L−1 of 1,3-1,6 β-glucans. Moreover, when the dystrophic larvae underwent 8 mg L−1 treatment, an improvement of the locomotor performances and mitochondrial respiration were observed. In conclusion, the observed results demonstrated that 1,3-1,6 β-glucans improve locomotor performances and mitochondrial function in dystrophic zebrafish. Therefore, for ameliorating their life quality, 1,3-1,6 β-glucans look like a promising diet supplement for DMD patients, even though further investigations are required.

The protective role of desmin in duchenne muscular dystrophy: Therapeutic insights.

The protective role of desmin in duchenne muscular dystrophy: Therapeutic insights.

Restorative treatments of dystrophin expression in Duchenne muscular dystrophy: A systematic review.

To evaluate the effect of pharmacological treatments that increase the synthesis of dystrophin in Duchenne muscular dystrophy (DMD). Systematic searches were carried out in MEDLINE, EMBASE, and Web of Science, and in gray literature from inception to December 2019. Clinical trials addressing the effect of restorative treatments of dystrophin expression in children and adolescents with DMD on functional outcomes {(6‐minute walking distance [6MWD], other timed functional tests [TFTs], The North Star Ambulatory Assessment)}, dystrophin expression, cardiorespiratory function, and biochemical tests were included. The DerSimonian‐Laird method was used to calculate the pooled estimates for functional outcomes. Eleven studies were included in the systematic review and five in the meta‐analysis. Eteplirsen showed a significant effect on 6MWD, Δ6MWD = 67.3 m (95% CI: 27.32, 107.28), and Δ6MWD = 151.0 m (95% CI: 36.15, 265.85) at 48 weeks and 3 years, respectively. In the systematic review, analyzing individually the clinical trials using Ataluren and Drisapersen showed a nonsignificant effect on 6MWD. However, the meta‐analysis showed a significant effect on 6MWD for Ataluren and Drisapersen, Δ6MWD = 18.3 m (95% CI: 1.0, 35.5) and Δ6MWD = 21.5 m (95% CI: 4.7, 38.3), respectively. There were no significant differences according to baseline age for Drisapersen. Similarly, the meta‐analysis showed effect in TFT with Ataluren. All drugs induced a partial synthesis of dystrophin, and exon skipping was obtained with Eteplirsen and Drisapersen. Eteplirsen also improved forced vital capacity (Δ%pFVC = 1.8%) and maximal inspiratory pressure (Δ%pMIP = 4.4%). Eteplirsen and Ataluren could modestly reduce disease progression. However, more trials are needed to confirm its efficacy, as well as quality of life and cost‐utility studies.

Restoration of Dystrophin in Duchenne Muscular Dystrophy by Human iPS Cells Derived Skeletal Muscle Progenitor Cells

Background and ObjectiveDuchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle‐wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) offer an important opportunity to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPCs) for DMD treatment.Methods and ResultshiPSCs were treated with CHIR99021(10 μM) for two days and followed by givinostat (100 nM) for 5 days in E6 medium (Fig. 1). After culture in E6 medium for additional seven days, cells were analyzed for myogenic makers (Pax7, desmin) expression. Reprogrammed cells (MPCs) from 4 human iPS cell lines expressed Pax7 and desmin. MPCs were differentiated into myotubes in N2 or DMEM medium with 2% horse serum expressing MF20. Additionally, we also generated MPCs from hiPSCs using CHIR99021 and FGF according to a previous study (Fig. 2) and referred these cells as control‐MPCs. We observed that givinostat induced MPCs exhibited superior proliferation and migration capacity using CCK‐8 assay, colony assay and migration assay compared to control‐MPCs. Moreover, we transplanted human myoblasts (1×105) and MPCs (1×105) labeled with GFP into tibialis anterior (TA) muscles of Mdx/scid mice, after injury with cardiotoxin (CTX) for one day prior to cell transplantation. One month after transplantation, dystrophin and GFP double positive fibers were analyzed. Givinostat induced MPCs showed higher engraftment capacity and restoration of dystrophin than treatment with control‐MPCs and human myoblasts. In addition, givinostat induced MPCs treated muscle contained significantly less inflammatory cells and showed limited muscle necrosis and fibrosis.ConclusionWe successfully generated highly expandable and integration free MPCs from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat induced MPCs showed marked and impressive regeneration and restored dystrophin in injured tibialis muscle compared to control MPCs. It is concluded that hiPSCs pharmacologically reprogrammed into MPCs with a small molecule, givinostat might be effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.Support or Funding InformationNational Institutes of Health grants R01 HL134354 & R01 AR070029 (M Ashraf, Y Tang, NL Weintraub)Schematic outline of generation of MPCs from hiPSCs using CHIR99021 and givinostat (Givi) small molecule combo.Figure 1Schematic outline generation of control MPCs from hiPSC.Figure 2

Cardiac Management of the Patient With Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) results in a progressive cardiomyopathy that produces significant morbidity and mortality. To improve the quality of life in patients with DMD, cardiac care is focused on surveillance and management, with the goal of slowing the onset and progression of heart failure complications. The current article is intended to be an expanded review on the cardiac management data used to inform the 2018 DMD Care Considerations recommendations as well as be a discussion on clinical controversies and future management directions. The new cardiac guidance includes changes regarding noninvasive imaging surveillance of cardiac function and pharmacologic therapy. Many emerging therapies lack sufficient evidence-based data to be recommended in the 2018 DMD Care Considerations. These are discussed in the present article as clinical controversies and future directions. Important emerging therapies include new heart failure medications, mechanical circulatory support with ventricular assist devices, heart transplantation, and internal cardiac defibrillators. Future research studies should be focused on the risks and benefits of these advanced therapies in patients with DMD. We conclude this review with a brief discussion on the relationship between the heart and the recently developed medications that are used to directly target the absence of dystrophin in DMD.

Nitric oxide-dependent attenuation of noradrenaline-induced vasoconstriction is impaired in the canine model of Duchenne muscular dystrophy.

We developed a novel method to study sympatholysis in dogs. We showed abolishment of sarcolemmal nNOS, and reduction of total nNOS and total eNOS in the canine Duchenne muscular dystrophy (DMD) model. We showed sympatholysis in dogs involving both nNOS-derived NO-dependent and NO-independent mechanisms. We showed that the loss of sarcolemmal nNOS compromised sympatholysis in the canine DMD model. We showed that NO-independent sympatholysis was not affected in the canine DMD model. The absence of dystrophin in Duchenne muscular dystrophy (DMD) leads to the delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma. Sarcolemmal nNOS plays an important role in sympatholysis, a process of attenuating reflex sympathetic vasoconstriction during exercise to ensure blood perfusion in working muscle. Delocalization of nNOS compromises sympatholysis resulting in functional ischaemia and muscle damage in DMD patients and mouse models. Little is known about the contribution of membrane-associated nNOS to blood flow regulation in dystrophin-deficient DMD dogs. We tested the hypothesis that the loss of sarcolemmal nNOS abolishes protective sympatholysis in contracting muscle of affected dogs. Haemodynamic responses to noradrenaline in the brachial artery were evaluated at rest and during contraction in the absence and presence of NOS inhibitors. We found sympatholysis was significantly compromised in DMD dogs, as well as in normal dogs treated with a selective nNOS inhibitor, suggesting that the absence of sarcolemmal nNOS underlies defective sympatholysis in the canine DMD model. Surprisingly, inhibition of all NOS isoforms did not completely abolish sympatholysis in normal dogs, suggesting sympatholysis in canine muscle also involves NO-independent mechanism(s). Our study established a foundation for using the dog model to test therapies aimed at restoring nNOS homeostasis in DMD.

Utrophin haploinsufficiency does not worsen the functional performance, resistance to eccentric contractions and force production of dystrophic mice.

The lack of dystrophin in Duchenne muscular dystrophy (DMD) compromises the integrity and function of muscle fibers. Skeletal muscles, except the diaphragm, do not undergo progressive degeneration in adult mdx mice due to compensatory mechanisms, including structural protein upregulation. New mouse models, including utrophin haploinsufficient mdx (mdx/utrn+/-) mice, may better recapitulate DMD. Our goal was to determine whether mdx/utrn+/- worsens the mdx phenotype and to characterize the course of the disease on muscle function and contractility at 1, 2, and 5 months of age, which encompass all stages of development relevant to DMD therapy. The functional performances of mdx/utrn+/- mice showed that they are not more affected than mdx/utrn+/+ mice based on downhill treadmill running parameters and subsequent recovery measured by open-field voluntary activity. WT mice ran the entire distance (450 m) on the treadmill, with an additional 561 m during the 4 h of open-field while mdx/utrn+/+ and mdx/utrn+/- mice completed, respectively, 236 m and 273 m on the treadmill and 341 m and 287 m during the open-field period. In addition, isolated ex vivo contractile properties and repeated eccentric contractions showed that mdx/utrn+/- does not significantly worsen the function of dystrophic EDL muscles, which are mainly composed of fast-twitch fibers that are preferentially affected in DMD. Twitch, absolute tetanic, and specific tetanic forces were very similar in dystrophic EDL muscles from mdx/utrn+/+ and mdx utrn+/- mice at 1, 2, and 5 months of age. Five-month-old mdx/utrn+/+ and mdx/utrn+/- mice lost roughly 50% of their force due to repeated eccentric contractions. Thus, histological, morphological, biochemical functional and contractile observations showed that utrophin haploinsufficiency has a very limited impact on mdx mice.

726. Prevention of Muscular Dystrophy by an AAV Vector Encoding a Nonimmunogenic Protein Based on the Evolution of Utrophin and Dystrophin

The majority of mutations causing Duchenne muscular dystrophy (DMD) are multi-exon, frameshifting deletions in the dystrophin gene. Expression of recombinant dystrophin in DMD therefore risks chronic immune recognition of the “non-self” protein. Early developmental expression of the paralogous protein utrophin in the thymus may confer central immunological tolerance to its peptide sequence. Here we address previously unanswered questions about the therapeutic efficacy and immunogenicity of “µutrophins” encoded by synthetic minigenes suitable for use in AAV. First we used a comparative phylogenomic approach to address a deceptively simple question with critical implications for this field: “which came first-dystrophin or the sarcomere”. This analysis provided evidence that the long rod-like domain of utrophin and dystrophin was “coopted” from a higher molecular weight protein in dynein-propelled animals lacking striated muscle. When considered in light of recent crystalographic studies, these results suggest that the full functionality of native dystrophin may be conferred by recombinant mini-utrophins with internally deleted rod domains that preserve inter-repeat folding and hence maximal tensile strength. We maximized recombinant µUtrophin (µU) expression from synthetic open reading frames by using the codon bias of striated myosin. Our blinded studies of mdx mice injected with a µU transgene are the first to show COMPLETE recovery of peripheral myofiber nucleation following a single systemic injection of an AAV vector. Sarcoglycan expression, as measured by western blot in the µU-treated mice, are restored to control levels throughout growth to skeletal maturity. To investigate the correlation between our histological findings and functionality, we validated a novel open field cage and attached running wheel system. Our blinded data show remarkable functional rescue in µU-treated mdx mice using this non-invasive test with relevance to the clinical assessment of disease progression in DMD. These data also strongly correlate with those derived from in vivo force grip and ex vivo force transducer measurements. Remarkably, a majority of mdx mice show complete normalization of serum creatine kinase (CK), a first for single-dose treatment by any clinically translatable modality. In dystrophic puppies, intravenous injection of a 30-fold lower relative dose of AAV9U fully restored sarcoglycan levels and normalized the myofiber size-distribution following a threefold increase in muscle mass. Interferon-gamma ELISpot assays using utrophin-derived peptides revealed no reactivity in injected dogs, consistent with central immunological tolerance. These findings suggest a rationale for neonatal gene therapy using utrophin as an internally deleted “self” protein in DMD to minimize the risk of chronic immunotoxicity. We outline a rigorous translational approach using escalating vector doses in GSHPMD dogs harboring a newly defined 5 megabase deletion encompassing the dystrophin ORF.

Applications of the PMO platform to genetic diseases

Genetic diseases are caused by a variety of mutations some of which lead to aberrant splicing of pre- mRNA and prevent proper mRNA translation of essential proteins or lead to translation of undesirable proteins. Such mRNA defects can be frequently repaired by appropriate targeting of oligonucleotides to modify splicing pathways and restore correct translation of desirable proteins. Hutchinson-Gilford progeria syndrome (HGPS), the main topic of this conference, other laminopathies, as well as diseases such as Duchenne muscular dystrophy (DMD) and thalassemia, are amenable to splicing manipulation or exon skipping. It has been shown in cell culture, in animal disease models or in case of DMD in clinical trials that oligonucleotides targeted to appropriate pre- mRNA splicing elements can restore correct splicing and allow production of desirable proteins, i.e. dystrophin in DMD, beta-globin in thalassemia, or reduce the level of harmful proteins, such as progerin in HGPS. Sarepta Therapeutics develops phosphorodiamidate morpholino oligomers (PMOs) and their derivatives as potential drugs for the treatment of rare diseases. After more than three years of treatment with PMO drug candidate, eteplirsen, stability of respiratory functions was observed and the results of the 6-minute walk test (6MWT) at 168 weeks showed continued ambulation across all patients evaluable on the test. Some decline in distance walked was observed since the week 144 time point. No significant treatment related adverse events were observed over the three-year course of this study.

A sensitive, reproducible and objective immunofluorescence analysis method of dystrophin in individual fibers in samples from patients with duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

What Can We Learn From Clinical Trials of Exon Skipping for DMD?

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK's Phase III clinical trial ({"type":"clinical-trial","attrs":{"text":"NCT01254019","term_id":"NCT01254019"}}NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to placebo.1 On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA) has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature.2 The news came as a great disappointment to the scientific community and more specifically to the families and foundations that had followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA's view of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching impact beyond exon skipping therapy in DMD.

P.11.13 Phosphorothioate modification increases capability of dystrophin exon 45 skipping and reduces cytotoxicity of RNA/ENA chimera

Antisense oligonucleotides (AOs)-mediated exon skipping is the most promising way to express internally deleted dystrophin in Duchenne muscular dystrophy (DMD) by correcting the reading frame of dystrophin mRNA. An antisense chimeric oligonucleotide consisting of 2-O-methyl RNA and ethylene-bridged nucleic acid (ENA) against exon 45 of the dystrophin gene, AO85, has been shown efficient in inducing exon 45 skipping. Phosphorothioate (PS)-modification is the most widely performed chemical modification of AO. Here, we introduced PS-modification to AO85 (AO88) and examined its exon skipping capability and cytotoxicity. The activity to induce exon 45 skipping was examined using primary muscle cells established from a DMD patient with a deletion of dystrophin exon 44. AO88 and AO85 were added to the culture medium and resulting dystrophin mRNAs were semiquantitated by a micro capillary electrophoresis of RT-PCR amplified products. AO88 was found to show activity to induce dystrophin exon 45 skipping from 50nM. More than 90% of splicing products lacked exon 45 at 400 nM. Remarkably, AO88 showed significantly higher exon skipping activity than AO85. Cytotoxicity assessed by the MTT Cell Proliferation Kit was lower in AO88 than AO85. In conclusion, PS-modification of RNA/ENA chimera was shown to render stronger exon skipping activity and lower cytotoxicity than PO-RNA/ENA chimera. It was assumed that AO88 has higher potential for clinical use than AO85.