Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by fibrotic tissue deposition in skeletal muscle. We assessed the role of periostin in fibrosis using mdx mice, an established DMD murine model, for which we conducted a thorough examination of periostin expression over a year. RNA and protein levels in diaphragm (DIA) muscles were assessed and complemented by a detailed histological analysis at 5 months of age. In dystrophic DIAs, periostin (Postn) mRNA expression significantly exceeded that seen in wildtype controls at all timepoints analysed, with the highest expression at 5 months of age (p < 0.05). We found Postn to be more consistently highly expressed at the earlier timepoints compared to established markers of fibrosis like transforming growth factor-beta 1 (Tgf-ß1) and connective tissue growth factor (Ctgf). Immunohistochemistry confirmed a significantly higher periostin protein expression in 5-month-old mdx mice compared to age-matched healthy controls (p < 0.01), coinciding with a significant fibrotic area percentage (p < 0.0001). RT-qPCR also indicated an elevated expression of Tgf-ß1, Col1α1 (collagen type 1 alpha 1) and Ctgf in mdx DIAs compared to wild type controls (p < 0.05) at 8- and 12-month timepoints. Accordingly, immunoblot quantification demonstrated elevated periostin (3, 5 and 8 months, p < 0.01) and Tgf-ß1 (8 and 12 months, p < 0.001) proteins in the mdx muscle. These findings collectively suggest that periostin expression is a valuable marker of fibrosis in this relevant model of DMD. They also suggest periostin as a potential contributor to fibrosis development, with an early onset of expression, thereby offering the potential for timely therapeutic intervention and its use as a biomarker in muscular dystrophies.

An Induced Pluripotent Stem Cell-Derived Human Blood-Brain Barrier (BBB) Model to Test the Crossing by Adeno-Associated Virus (AAV) Vectors and Antisense Oligonucleotides.

The blood-brain barrier (BBB) is the specialised microvasculature system that shields the central nervous system (CNS) from potentially toxic agents. Attempts to develop therapeutic agents targeting the CNS have been hindered by the lack of predictive models of BBB crossing. In vitro models mimicking the human BBB are of great interest, and advances in induced pluripotent stem cell (iPSC) technologies and the availability of reproducible differentiation protocols have facilitated progress. In this study, we present the efficient differentiation of three different wild-type iPSC lines into brain microvascular endothelial cells (BMECs). Once differentiated, cells displayed several features of BMECs and exhibited significant barrier tightness as measured by trans-endothelial electrical resistance (TEER), ranging from 1500 to >6000 Ωcm2. To assess the functionality of our BBB models, we analysed the crossing efficiency of adeno-associated virus (AAV) vectors and peptide-conjugated antisense oligonucleotides, both currently used in genetic approaches for the treatment of rare diseases. We demonstrated superior barrier crossing by AAV serotype 9 compared to serotype 8, and no crossing by a cell-penetrating peptide-conjugated antisense oligonucleotide. In conclusion, our study shows that iPSC-based models of the human BBB display robust phenotypes and could be used to screen drugs for CNS penetration in culture.

Novel Metabolomic Approach for Identifying Pathology-Specific Biomarkers in Rare Diseases: A Case Study in Oculopharyngeal Muscular Dystrophy (OPMD).

The identification of metabolomic biomarkers relies on the analysis of large cohorts of patients compared to healthy controls followed by the validation of markers in an independent sample set. Indeed, circulating biomarkers should be causally linked to pathology to ensure that changes in the marker precede changes in the disease. However, this approach becomes unfeasible in rare diseases due to the paucity of samples, necessitating the development of new methods for biomarker identification. The present study describes a novel approach that combines samples from both mouse models and human patients to identify biomarkers of OPMD. We initially identified a pathology-specific metabolic fingerprint in murine dystrophic muscle. This metabolic fingerprint was then translated into (paired) murine serum samples and then to human plasma samples. This study identified a panel of nine candidate biomarkers that could predict muscle pathology with a sensitivity of 74.3% and specificity of 100% in a random forest model. These findings demonstrate that the proposed approach can identify biomarkers with good predictive performance and a higher degree of confidence in their relevance to pathology than markers identified in a small cohort of human samples alone. Therefore, this approach has a high potential utility for identifying circulating biomarkers in rare diseases.

Microdystrophin Gene Addition Significantly Improves Muscle Functionality and Diaphragm Muscle Histopathology in a Fibrotic Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease affecting 1:5000 newborn males. No cure is currently available, but gene addition therapy, based on the adeno-associated viral (AAV) vector-mediated delivery of microdystrophin transgenes, is currently being tested in clinical trials. The muscles of DMD boys present significant fibrotic and adipogenic tissue deposition at the time the treatment starts. The presence of fibrosis not only worsens the disease pathology, but also diminishes the efficacy of gene therapy treatments. To gain an understanding of the efficacy of AAV-based microdystrophin gene addition in a relevant, fibrotic animal model of DMD, we conducted a systemic study in juvenile D2.mdx mice using the single intravenous administration of an AAV8 system expressing a sequence-optimized murine microdystrophin, named MD1 (AAV8-MD1). We mainly focused our study on the diaphragm, a respiratory muscle that is crucial for DMD pathology and that has never been analyzed after treatment with AAV-microdystrophin in this mouse model. We provide strong evidence here that the delivery of AAV8-MD1 provides significant improvement in body-wide muscle function. This is associated with the protection of the hindlimb muscle from contraction-induced damage and the prevention of fibrosis deposition in the diaphragm muscle. Our work corroborates the observation that the administration of gene therapy in DMD is beneficial in preventing muscle fibrosis.

Systemic Delivery of a Monoclonal Antibody to Immunologically Block Myostatin in the A17 Mouse Model of OPMD.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset rare muscle disease affecting approximately 1 in 80,000 individuals worldwide. However, it can affect as much as 1:600 individuals in some populations due to a strong founder effect. The muscle pathology is characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia), and limb weakness at later stages of disease progression. The genetic defect is associated with significant fibrotic deposition and atrophy in affected muscles. No treatments are available to cure the disease. Only surgical techniques to correct ptosis and swallowing are currently possible, though they carry a risk of recurrence. Myostatin is a negative regulator of muscle growth, and several strategies to downregulate its expression have been developed with the aim of improving muscle mass and strength in muscular pathologies. We recently showed that weekly systemic treatment of the A17 murine model of OPMD with a monoclonal antibody for myostatin improves body and muscle mass, increases muscle strength, and reduces muscle fibrosis. Here, we describe the methodology for repeated intraperitoneal delivery of myostatin antibody in the murine model. Furthermore, we detail the most relevant analyses to assess histopathological and functional improvements of this treatment in this mouse model.

Long-Term Systemic Treatment of a Mouse Model Displaying Chronic FSHD-like Pathology with Antisense Therapeutics That Inhibit DUX4 Expression.

Silencing the expression of the double homeobox 4 (DUX4) gene offers great potential for the treatment of facioscapulohumeral muscular dystrophy (FSHD). Several research groups have recently reported promising results using systemic antisense therapy in a transgenic small animal model of FSHD, the ACTA1-MCM/FLExDUX4 mouse model. However, the treatment was applied in non-DUX4-induced mice or shortly after DUX4 activation, which resulted in conditions that do not correctly represent the situation in a clinic. Here, we generated progressive FSHD-like pathology in ACTA1-MCM/FLExDUX4 mice and then treated the animals with vivoPMO-PACS4, an antisense compound that efficiently downregulates DUX4. To best mimic the translation of this treatment in clinical settings, the systemic antisense oligonucleotide administration was delayed to 3 weeks after the DUX4 activation so that the pathology was established at the time of the treatment. The chronic administration of vivoPMO-PACS4 for 8 weeks downregulated the DUX4 expression by 60%. Consequently, the treated mice showed an increase by 18% in body-wide muscle mass and 32% in muscle strength, and a reduction in both myofiber central nucleation and muscle fibrosis by up to 29% and 37%, respectively. Our results in a more suitable model of FSHD pathology confirm the efficacy of vivoPMO-PACS4 administration, and highlight the significant benefit provided by the long-term treatment of the disease.

Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 5000 newborn boys. It is caused by mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibers and their replacement with fibro-adipogenic tissue. Using antisense oligonucleotides (AONs) to modify out-of-frame mutations in the DMD gene, named exon skipping, is currently considered among the most promising treatments for DMD patients. The development of this strategy is rapidly moving forward, and AONs designed to skip exons 51 and 53 have received accelerated approval in the USA. In preclinical setting, the mdx mouse model, carrying a point mutation in exon 23 of the murine Dmd gene that prevents production of dystrophin protein, has emerged as a valuable tool, and it is widely used to study in vivo therapeutic approaches for DMD. Here we describe the methodology for intravenous delivery of AONs targeting dystrophin through tail vein of mdx mice. Furthermore, the most relevant functional analyses to be performed in living mice, and the most informative histopathological and molecular assays to evaluate the effect of this treatment are detailed.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

Improving Molecular and Histopathology in Diaphragm Muscle of the Double Transgenic ACTA1-MCM/FLExDUX4 Mouse Model of FSHD with Systemic Antisense Therapy.

Facioscapulohumeral muscular dystrophy (FSHD) is a rare muscle dystrophy causing muscle weakness initially in the face, shoulders, and upper arms, and extends to lower body muscles as the disease progresses. Respiratory restriction in FSHD is increasingly reported to be more common and severe than previously thought, with the involvement of diaphragm weakness in pulmonary insufficiency being under debate. As aberrant expression of the double homeobox 4 (DUX4) gene is the prime cause of FSHD, we and others have developed numerous strategies and reported promising results on downregulating DUX4 expression in both cellular and animal models of FSHD. However, the effect of DUX4 and anti-DUX4 approaches on diaphragm muscle has not been elucidated. In this study, we show that toxic DUX4 expression causes pathology that affects the diaphragm of ACTA1-MCM/FLExDUX4 mouse model of FSHD at both molecular and histological levels. Of importance, a systemic antisense treatment that suppresses DUX4 and target genes expression by 50% significantly improves muscle regeneration and muscle fibrosis, and prevents modification in myofiber type composition, supporting its development as a treatment for FSHD.

Knockdown of Muscle-Specific Ribosomal Protein L3-Like Enhances Muscle Function in Healthy and Dystrophic Mice.

Ribosomal protein L3-like (RPL3L) is a poorly characterized ribosomal protein that is exclusively expressed in skeletal and cardiac muscle. RPL3L is also downregulated in Duchenne muscular dystrophy (DMD), suggesting that it may play an important role in muscle biology. In this study, we investigated the role of RPL3L in skeletal muscle of healthy C57 and dystrophic mdx mice. We show that RPL3L is developmentally regulated and that intramuscular adeno-associated virus (AAV)-mediated RPL3L knockdown in the tibialis anterior of C57 and mdx mice results in increased specific force with improved resistance to eccentric contraction induced muscle damage in dystrophic muscles. The mechanism by which RPL3L knockdown improves muscle function remains unclear. Histological observations showed a significant increase in muscle length and decrease in muscle cross-sectional area after RPL3L inhibition suggesting that this ribosomal protein may play a role in myofiber morphology. The endogenous downregulation of RPL3L in DMD may be a protective mechanism that attempts to improve skeletal muscle function and counteract the dystrophic phenotype.

Systemic antisense therapeutics inhibiting DUX4 expression ameliorates FSHD-like pathology in an FSHD mouse model.

Aberrant expression of the double homeobox 4 (DUX4) gene in skeletal muscle causes muscle deterioration and weakness in Facioscapulohumeral muscular dystrophy (FSHD). Since the presence of a permissive pLAM1 polyadenylation signal is essential for stabilization of DUX4 mRNA and translation of DUX4 protein, disrupting the function of this structure can prevent expression of DUX4. We and others have shown promising results using antisense approaches to reduce DUX4 expression in vitro and in vivo following local intramuscular administration. Here we demonstrate that further development of the antisense chemistries enhances in vitro antisense efficacy. The optimal chemistry was conjugated to a cell-penetrating moiety and was systemically administered into the tamoxifen-inducible Cre-driver FLExDUX4 double-transgenic mouse model of FSHD. After four weekly treatments, mRNA quantities of DUX4 and target genes were reduced by 50% that led to 12% amelioration in muscle atrophy, 52% improvement in in situ muscle strength, 17% reduction in muscle fibrosis and prevention of shift in the myofiber type profile. Systemic DUX4 inhibition also significantly improved the locomotor activity and reduced the fatigue level by 22%. Our data demonstrate that the optimized antisense approach has potential of being further developed as a therapeutic strategy for FSHD.

Dose-Dependent Microdystrophin Expression Enhancement in Cardiac Muscle by a Cardiac-Specific Regulatory Element.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease that affects 1:5,000 live male births and is characterized by muscle wasting. By the age of 13 years, affected individuals are often wheelchair bound and suffer from respiratory and cardiac failure, which results in premature death. Although the administration of corticosteroids and ventilation can relieve the symptoms and extend the patients' lifespan, currently no cure exists for DMD. Among the different approaches under preclinical and clinical testing, gene therapy, using adeno-associated viral (AAV) vectors, is one of the most promising. In this study, we delivered intravenously AAV9 vectors expressing the microdystrophin MD1 (ΔR4-R23/ΔCT) under control of the synthetic muscle-specific promoter Spc5-12 and assessed the effect of adding a cardiac-specific cis-regulatory module (designated as CS-CRM4) on its expression profile in skeletal and cardiac muscles. Results show that Spc5-12 promoter, in combination with an AAV serotype that has high tropism for the heart, drives high MD1 expression levels in cardiac muscle in mdx mice. The additional regulatory element CS-CRM4 can further improve MD1 expression in cardiac muscles, but its effect is dose dependent and enhancement becomes evident only at lower vector doses.

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.

Researcher's Perceptions on Publishing "Negative" Results and Open Access.

Scientific advance is based on reproducibility, corroboration, and availability of research results. However, large numbers of experimental results that contradict previous work do not get published and many research results are not freely available as they are hidden behind paywalls. As part of COST Action "DARTER", a network of researchers in the field of RNA therapeutics, we have performed a small survey among our members and their colleagues to assess their opinion on the subject of publishing contradictory or ambiguous results and their attitude to open access (OA) publishing. Our survey indicates that, although researchers highly value publication of "negative" results, they often do not publish their own, citing lack of time and the perception that those results may not be as highly cited. OA, on the other hand, seems to be widely accepted, but in many cases not actively sought by researchers due to higher costs associated with it.

Inhibition of Myostatin Reduces Collagen Deposition in a Mouse Model of Oculopharyngeal Muscular Dystrophy (OPMD) With Established Disease.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease presented by ptosis, dysphagia, and limb weakness. Affected muscles display increased fibrosis and atrophy, with characteristic inclusion bodies in the nucleus. Myostatin is a negative regulator of muscle mass, and inhibition of myostatin has been demonstrated to improve symptoms in models of muscular dystrophy. METHODS: We systemically administered a monoclonal antibody to block myostatin in the A17 mouse model of OPMD at 42 weeks of age. The mice were administered a weekly dose of 10 mg/kg RK35 intraperitonially for 10 weeks, following which serum and histological analyses were performed on muscle samples. RESULTS: The administration of the antibody resulted in a significant decrease in serum myostatin and collagen deposition in muscles. However, minimal effects on body mass, muscle mass and myofiber diameter, or the density of intranuclear inclusions (INIs) (a hallmark of disease progression of OPMD) were observed. CONCLUSION: This study demonstrates that inhibition of myostatin does not revert muscle atrophy in a mouse model with established OPMD disease, but is effective at reducing observed histological markers of fibrosis in the treated muscles.

Myostatin inhibition in combination with antisense oligonucleotide therapy improves outcomes in spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by genetic defects in the survival motor neuron 1 (SMN1) gene that lead to SMN deficiency. Different SMN-restoring therapies substantially prolong survival and function in transgenic mice of SMA. However, these therapies do not entirely prevent muscle atrophy and restore function completely. To further improve the outcome, we explored the potential of a combinatorial therapy by modulating SMN production and muscle-enhancing approach as a novel therapeutic strategy for SMA. METHODS: The experiments were performed in a mouse model of severe SMA. A previously reported 25-mer morpholino antisense oligomer PMO25 was used to restore SMN expression. The adeno-associated virus-mediated expression of myostatin propeptide was used to block the myostatin pathway. Newborn SMA mice were treated with a single subcutaneous injection of 40 µg/g (therapeutic dose) or 10 µg/g (low-dose) PMO25 on its own or together with systemic delivery of a single dose of adeno-associated virus-mediated expression of myostatin propeptide. The multiple effects of myostatin inhibition on survival, skeletal muscle phenotype, motor function, neuromuscular junction maturation, and proprioceptive afferences were evaluated. RESULTS: We show that myostatin inhibition acts synergistically with SMN-restoring antisense therapy in SMA mice treated with the higher therapeutic dose PMO25 (40 µg/g), by increasing not only body weight (21% increase in male mice at Day 40), muscle mass (38% increase), and fibre size (35% increase in tibialis anterior muscle in 3 month female SMA mice), but also motor function and physical performance as measured in hanging wire test (two-fold increase in time score) and treadmill exercise test (two-fold increase in running distance). In SMA mice treated with low-dose PMO25 (10 µg/g), the early application of myostatin inhibition prolongs survival (40% increase), improves neuromuscular junction maturation (50% increase) and innervation (30% increase), and increases both the size of sensory neurons in dorsal root ganglia (60% increase) and the preservation of proprioceptive synapses in the spinal cord (30% increase). CONCLUSIONS: These data suggest that myostatin inhibition, in addition to the well-known effect on muscle mass, can also positively influence the sensory neural circuits that may enhance motor neurons function. While the availability of the antisense drug Spinraza for SMA and other SMN-enhancing therapies has provided unprecedented improvement in SMA patients, there are still unmet needs in these patients. Our study provides further rationale for considering myostatin inhibitors as a therapeutic intervention in SMA patients, in combination with SMN-restoring drugs.

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500-5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

Inhibition of myostatin improves muscle atrophy in oculopharyngeal muscular dystrophy (OPMD).

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease affecting one per 80 000 of the general population characterized by profound dysphagia and ptosis, and limb weakness at later stages. Affected muscles are characterized by increased fibrosis and atrophy. Myostatin is a negative regulator of muscle mass, and inhibition of myostatin has been demonstrated to ameliorate symptoms in dystrophic muscles. METHODS: In this study, we performed a systemic delivery of a monoclonal antibody to immunologically block myostatin in the A17 mouse model of OPMD. The mice were administered a weekly dose of 10 mg/kg RK35 intraperitonially for 10 weeks, following which histological analyses were performed on the samples. RESULTS: This treatment significantly (P < 0.01) improved body mass (11%) and muscle mass (for the tibialis anterior and extensor digitorum longus by 19% and 41%) in the A17 mice treated with RK35 when compared to saline controls. Similarly, a significantly (P < 0.01) increased muscle strength (18% increase in maximal tetanic force) and myofibre diameter (17% and 44% for the tibialis anterior and extensor digitorum longus), and reduced expression of markers of muscle fibrosis (40% reduction in area of expression), was also observed. No change in the density of intranuclear inclusions (a hallmark of disease progression of OPMD) was however observed. CONCLUSIONS: Our study supports the clinical translation of such antibody-mediated inhibition of myostatin as a treatment of OPMD. This strategy has implications to be used as adjuvant therapies with gene therapy based approaches, or to stabilize the muscle prior to myoblast transplantation.

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.