Activin A-Expressing Polymorphonuclear Myeloid-Derived Suppressor Cells Infiltrate Skeletal and Cardiac Muscle and Promote Cancer Cachexia.

Cachexia is a major cause of death in cancer and leads to wasting of cardiac and skeletal muscle, as well as adipose tissue. Various cellular and soluble mediators have been postulated in driving cachexia; however, the specific mechanisms behind this muscle wasting remain poorly understood. In this study, we found polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) to be critical for the development of cancer-associated cachexia. Significant expansion of PMN-MDSCs was observed in the cardiac and skeletal muscles of cachectic murine models. Importantly, the depletion of this cell subset, using depleting anti-Ly6G Abs, attenuated this cachectic phenotype. To elucidate the mechanistic involvement of PMN-MDSCs in cachexia, we examined major mediators, that is, IL-6, TNF-α, and arginase 1. By employing a PMN-MDSC-specific Cre-recombinase mouse model, we showed that PMN-MDSCs were not maintained by IL-6 signaling. In addition, PMN-MDSC-mediated cardiac and skeletal muscle loss was not abrogated by deficiency in TNF-α or arginase 1. Alternatively, we found PMN-MDSCs to be critical producers of activin A in cachexia, which was noticeably elevated in cachectic murine serum. Moreover, inhibition of the activin A signaling pathway completely protected against cardiac and skeletal muscle loss. Collectively, we demonstrate that PMN-MDSCs are active producers of activin A, which in turn induces cachectic muscle loss. Targeting this immune/hormonal axis will allow the development of novel therapeutic interventions for patients afflicted with this debilitating syndrome.

Development of DG9 peptide-conjugated single- and multi-exon skipping therapies for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.

DUX4 Transcript Knockdown with Antisense 2'-O-Methoxyethyl Gapmers for the Treatment of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder characterized by a progressive, asymmetric weakening of muscles, starting with those in the upper body. It is caused by aberrant expression of the double homeobox protein 4 gene (DUX4) in skeletal muscle. FSHD is currently incurable. We propose to develop a therapy for FSHD using antisense 2'-O-methoxyethyl (2'-MOE) gapmers, to knock down DUX4 mRNA expression. Using immortalized patient-derived muscle cells and local intramuscular injections in the FLExDUX4 FSHD mouse model, we showed that our designed 2'-MOE gapmers significantly reduced DUX4 transcript levels in vitro and in vivo, respectively. Furthermore, in vitro, we observed significantly reduced expression of DUX4-activated downstream targets, restoration of FSHD signature genes by RNA sequencing, significant improvements in myotube morphology, and minimal off-target activity. This work facilitates the development of a promising candidate therapy for FSHD and lays down the foundation for in vivo systemic treatment studies.

Optimization of antisense-mediated exon skipping for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is one of the most common lethal muscle-wasting disorders affecting young boys caused by mutations in the DMD gene. Exon skipping has emerged as a promising therapy for DMD. Antisense oligonucleotides (AONs) are designed to induce the skipping of exon(s), in order to restore the reading frame, and therefore, allow for dystrophin expression. Eteplirsen and golodirsen, AONs for DMD exons 51 and 53 skipping, have been recently approved by the FDA. Viltolarsen, an AON for DMD exon 53 skipping, was approved in Japan earlier this year. Although promising, the efficacy of eteplirsen and AON sequence employed remain controversial. In addition, exon skipping faces challenges including the applicability and delivery. This article reviews and discusses exon skipping and the current advances being made in the field, on drugs, multi-exon skipping, sequence design, and applicability. We also discuss challenges and future directions that will facilitate the development of exon skipping therapy.

CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.