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.

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.

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.

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.

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.

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-β1) 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-β1, 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-β1 (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.