COULIS, G. | Search bioRxiv

bioRxiv2025

Sustained efficacy of CRISPR-Cas13b gene therapy for FSHD is challenged by immune response to Cas13b

Facioscapulohumeral muscular dystrophy (FSHD) is a potentially devastating muscle disease caused by de-repression of the toxic DUX4 gene in skeletal muscle. FSHD patients may benefit from DUX4 inhibition therapies, and although several experimental strategies to reduce DUX4 levels in skeletal muscle are being developed, no approved disease modifying therapies currently exist. We developed a CRISPR-Cas13b system that cleaves DUX4 mRNA and reduces DUX4 protein level, protects cells from DUX4-mediated death, and reduces FSHD-associated biomarkers in vitro. In vivo delivery of the CRISPR-Cas13b system with adeno-associated viral vectors reduced acute damage caused by high DUX4 levels in a mouse model of severe FSHD. However, protection was not sustained over time, with decreases in Cas13b and guide RNA levels between 8 weeks and 6 months after injection. In addition, wild-type mice injected with AAV6.Cas13b showed muscle inflammation with infiltrates containing Cas13b-responsive CD8+ cytotoxic T cells. Our RNA-seq data confirmed that several immune response pathways were significantly increased in human FSHD myoblasts transfected with Cas13b. Overall, our findings suggest that CRISPR-Cas13b is highly effective for DUX4 silencing but successful implementation of CRISPR/Cas13-based gene therapies may require strategies to mitigate immune responses.

molecular biology↗

bioRxiv2023

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3+ macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3+ macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

immunology↗