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Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-β1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-β1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-β1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications. [ABSTRACT FROM AUTHOR]

Aberrant expression of the double homeobox 4 ( DUX4 ) gene in skeletal muscle predominantly drives the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). We recently demonstrated that berberine, an herbal extract known for its ability to stabilize guanine-quadruplex structures, effectively downregulates DUX4 expression in FSHD patient-derived myoblasts and in mice overexpressing exogenous DUX4 after viral vector-based treatment. Here, we sought to confirm berberine's inhibitory efficacy on DUX4 in the widely used FSHD-like transgenic mouse model, ACTA1-MCM/FLExDUX4, where DUX4 is induced at pathogenic levels using tamoxifen. Animals repeatedly treated with berberine via intraperitoneal injections for 4 weeks exhibited significant reductions in both mRNA and protein levels of DUX4 , and in mRNA expression of murine DUX4-related genes. This inhibition translated into improved forelimb muscle strength and positive alterations in important FSHD-relevant cellular pathways, although its impact on muscle mass and histopathology was less pronounced. Collectively, our data confirm the efficacy of berberine in downregulating DUX4 expression in the most relevant FSHD mouse model. However, further optimization of dosing regimens and new studies to enhance the bioavailability of berberine in skeletal muscle are warranted to fully leverage its therapeutic potential for FSHD 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., (© 2022. The Author(s).)

With the recent conditional approval of an antisense oligonucleotide (AON) that restores the reading frame of DMD transcript in a subset of Duchenne muscular dystrophy patients, it has been established that AONs sharing similar chemistry have clear clinical potential. Genetic diseases, such as facioscapulohumeral dystrophy (FSHD), can be the result of gain-of-function mutations. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3'UTR elements, it follows that targeting these elements with AONs have the potential to induce gene silencing. Aberrant expression of the Double homeobox 4 (DUX4) transcription factor and the downstream consequences of such expression is the hallmark of FSHD. Here we describe the bioinformatic strategies behind the design of AONs targeting polyadenylation signals and the methodologies relevant to their in vitro screening for efficacy and safety, including analysis of expression at the transcript and protein level of the specific target and downstream genes, and measurement of the effect on the fusion index of myotubes. The targeting of permissive DUX4 and MSTN are used as examples. MSTN encodes for myostatin, a negative regulator of myogenesis; the downregulation of MSTN expression has the potential to address the muscular atrophy associated with muscular dystrophies, sarcopenia, cancer and acquired immunodeficiency syndrome.

Using antisense oligonucleotides (AOs) to reframe mutated dystrophin, a recently developed therapeutic approach for Duchenne muscular dystrophy (DMD) named exon skipping, is considered among the most promising treatments for DMD patients. The development of this strategy is rapidly moving forward and the AO designed to skip exon 51 has received accelerated approval in the USA. However the strong complexity of the DMD pathology suggests that at least in older patients, where the muscle structure is almost completely compromised and the muscle is wasted and significantly infiltrated with fat and connective tissue, combined therapeutic approaches should be developed to approach the disease more effectively. Here we describe the methodology for the systemic intravenous delivery of AOs targeting dystrophin and myostatin in mdx mice, a DMD mouse model, in order to express dystrophin while downregulating myostatin, aiming for an increase in the muscle size and muscle strength. 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.