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

ABSTRACT: 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.

SUBMITTER: Lu-Nguyen N

PROVIDER: S-EPMC9313434 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

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Designed U7 snRNAs inhibit <i>DUX4</i> expression and improve FSHD-associated outcomes in <i>DUX4</i> overexpressing cells and FSHD patient myotubes.

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Project description:Facioscapulohumeral muscular dystrophy (FSHD) is a common, dominantly inherited disease caused by the epigenetic de-repression of the DUX4 gene, a transcription factor normally repressed in skeletal muscle. As targeted therapies are now possible in FSHD, a better understanding of the relationship between DUX4 activity, muscle pathology and muscle magnetic resonance imaging (MRI) changes is crucial both to understand disease mechanisms and for the design of future clinical trials. Here, we performed MRIs of the lower extremities in 36 individuals with FSHD, followed by needle muscle biopsies in safely accessible muscles. We examined the correlation between MRI characteristics, muscle pathology and expression of DUX4 target genes. Results show that the presence of elevated MRI short tau inversion recovery signal has substantial predictive value in identifying muscles with active disease as determined by histopathology and DUX4 target gene expression. In addition, DUX4 target gene expression was detected only in FSHD-affected muscles and not in control muscles. These results support the use of MRI to identify FSHD muscles most likely to have active disease and higher levels of DUX4 target gene expression and might be useful in early phase therapeutic trials to demonstrate target engagement in therapies aiming to suppress DUX4 expression.

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Therapeutic Strategies Targeting DUX4 in FSHD.

Project description:Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle dystrophy typically affecting patients within their second decade. Patients initially exhibit asymmetric facial and humeral muscle damage, followed by lower body muscle involvement. FSHD is associated with a derepression of DUX4 gene encoded by the D4Z4 macrosatellite located on the subtelomeric part of chromosome 4. DUX4 is a highly regulated transcription factor and its expression in skeletal muscle contributes to multiple cellular toxicities and pathologies ultimately leading to muscle weakness and atrophy. Since the discovery of the FSHD candidate gene DUX4, many cell and animal models have been designed for therapeutic approaches and clinical trials. Today there is no treatment available for FSHD patients and therapeutic strategies targeting DUX4 toxicity in skeletal muscle are being actively investigated. In this review, we will discuss different research areas that are currently being considered to alter DUX4 expression and toxicity in muscle tissue and the cell and animal models designed to date.

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Systemic Antisense Therapeutics for Dystrophin and Myostatin Exon Splice Modulation Improve Muscle Pathology of Adult mdx Mice.

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Project description:Aberrant expression of the full-length isoform of DUX4 (DUX4-FL) appears to underlie pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). DUX4-FL is a transcription factor and ectopic expression of DUX4-FL is toxic to most cells. Previous studies showed that DUX4-FL-induced pathology requires intact homeodomains and that transcriptional activation required the C-terminal region. In this study, we further examined the functional domains of DUX4 by generating mutant, deletion, and fusion variants of DUX4. We compared each construct to DUX4-FL for (i) activation of a DUX4 promoter reporter, (ii) expression of the DUX4-FL target gene ZSCAN4, (iii) effect on cell viability, (iv) activation of endogenous caspases, and (v) level of protein ubiquitination. Each construct produced a similarly sized effect (or lack of effect) in each assay. Thus, the ability to activate transcription determined the extent of change in multiple molecular and cellular properties that may be relevant to FSHD pathology. Transcriptional activity was mediated by the C-terminal 80 amino acids of DUX4-FL, with most activity located in the C-terminal 20 amino acids. We also found that non-toxic constructs with both homeodomains intact could act as inhibitors of DUX4-FL transcriptional activation, likely due to competition for promoter sites.This article has an associated First Person interview with the first author of the paper.

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Systemic delivery of a DUX4-targeting antisense oligonucleotide to treat facioscapulohumeral muscular dystrophy.

Project description:Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent skeletal muscle dystrophies. Skeletal muscle pathology in individuals with FSHD is caused by inappropriate expression of the transcription factor DUX4, which activates different myotoxic pathways. At the moment there is no molecular therapy that can delay or prevent skeletal muscle wasting in FSHD. In this study, a systemically delivered antisense oligonucleotide (ASO) targeting the DUX4 transcript was tested in vivo in ACTA1-MCM;FLExDUX4 mice that express DUX4 in skeletal muscles. We show that the DUX4 ASO was well tolerated and repressed the DUX4 transcript, DUX4 protein, and mouse DUX4 target gene expression in skeletal muscles. In addition, the DUX4 ASO alleviated the severity of skeletal muscle pathology and partially prevented the dysregulation of inflammatory and extracellular matrix genes. DUX4 ASO-treated ACTA1-MCM;FLExDUX4 mice performed better on a treadmill; however, the hanging grid and four-limb grip strength tests were not improved compared to control ASO-treated ACTA1-MCM;FLExDUX4 mice. This study shows that systemic delivery of ASOs targeting DUX4 is a promising therapeutic strategy for FSHD and strategies that further improve the ASO efficacy in skeletal muscle are warranted.

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Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

Project description:Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

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