Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common dominant muscular dystrophies and remains without an approved disease modifying therapy. Caused by the aberrant expression of the cytotoxic gene DUX4, FSHD is typically diagnosed in adulthood, however clinical onset in children (<18 years of age) is often associated with a more severe and rapid disease. While clinical trials are underway, a lack of human-specific pre-clinical models limit effective testing of potential therapies, particularly in children. To fill this gap, we describe here the development of induced pluripotent stem cell-derived 2-and 3-dimensional skeletal muscle models of children with clinically defined mild, moderate, and severe FSHD. These iPSC-derived muscle models replicate key features of FSHD, including aberrant DUX4 mRNA expression, muscle atrophy, and weakness, which correlate with the individuals’ specific disease severity. Next, we assessed the efficacy of adenine base editing (ABE) as a potential gene therapy approach to treat FSHD. DUX4-targeted ABE reduced DUX4 mRNA expression, improved muscle area and force generation in the most severe individual. Together this work supports the use of iPSC-derived skeletal muscle models as a less invasive method to study childhood-onset FSHD and establishes targeted DUX4 gene editing therapies as a potential treatment approach.
LMNArelated congenital muscular dystrophy (L-CMD) is one of the most severe laminopathies, which are incurable diseases primarily caused by pathogenic LMNA variants. LMNA encodes Lamin A/C: key components of the nuclear lamina, which provides structural stability to the nucleus, whilst regulating chromatin organisation and gene expression. L-CMD research is hindered by lack of humanised, tissue-specific models that accurately recapitulate disease phenotypes. We previously reported nuclear shape abnormalities and Lamin mislocalisation in LMNA-mutant induced pluripotent stem cell (iPSC)-derived skeletal muscle cells. Here, we expand the selection of L-CMD iPSCs, validate disease-associated readouts using a transgene-free differentiation protocol and assess gene editing strategies using 2D and 3D cultures. Results showed no overt defects in developmental myogenesis but recapitulated pathological nuclear shape abnormalities in monolayer cultures and engineered muscles, nuclear envelope protein mislocalisation and transcriptomic alterations across multiple pathogenic LMNA variants. We then used our platform to assess outcomes of LMNA gene editing. CRISPR-based exon-removal generated sRNA and protein Lamin A/C species, without normalisation of nuclear morphology or transcriptomic profile. Conversely, precise editing of the same variant corrected nuclear morphometrics, alongside normalisation of the pro-inflammatory transcriptomic signature, providing an advanced, humanised platform for translational research and precision medicine in laminopathies.
D. Moore, H. Steele-Stallard, L. Pinton et al.· Molecular Therapy· 0 citations
Myotonic dystrophy type 1 (DM1) is caused by an expansion of a CTG repeat in the 3′ untranslated region of the DMPK gene, leading to accumulation of toxic CUG-repeat RNAs, sequestration of MBNL proteins and widespread splicing dysregulation. Congenital DM1 (CDM), the most severe form of the disease, is associated with profound muscular and neurodevelopmental defects, yet the mechanisms underlying early human brain involvement remain poorly understood. Here, we generated cortical organoids from patient-derived pluripotent stem cells carrying >1000 CTG repeats, an expansion typically associated with CDM, to model early human neurodevelopment. DM1 molecular and cellular hallmarks were detected at early developmental stages, including nuclear DMPK RNA foci in neural progenitor cells and reduced proliferative capacity. As organoids matured, CDM cultures displayed altered cortical composition, with reduced CTIP2⁺ and SATB2⁺ neuronal populations and increased NFIA⁺/GFAP⁺ glial cells. In parallel, 120-day-old organoids recapitulated splicing abnormalities previously identified in DM1 patient brain tissue. To assess the contribution of MBNL dysfunction, we analyzed cortical organoids derived from MBNL2 and MBNL1/2/3 knockout induced pluripotent stem cells, which reproduced key neurodevelopmental phenotypes observed in CDM organoids, supporting a central role for MBNL loss of function in impaired corticogenesis. Finally, we evaluated the translational relevance of this model using tideglusib and erythromycin, two compounds currently under clinical evaluation in DM1 patients. Both treatments reduced DMPK RNA foci and restored proliferation defects in SOX2⁺ neural progenitors. Together, these findings establish cortical organoids as a robust human model of CDM-associated neurodevelopmental defects, uncover MBNL-dependent mechanisms underlying early corticogenesis impairment and demonstrate the utility of this platform for translational therapeutic discovery in DM1.
Azania Abatan, Jérôme Polentes, M. Bouquier et al.· bioRxiv· 0 citations
It is shown that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells, while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone, with a similar trend observed in C2C12 myoblasts.
H. Hoffmann, A. Finkelstein, Amanuel W. Geremew et al.· bioRxiv· 0 citations
Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder caused by mutations in the DMD gene, with deletions within the exon 45–55 hotspot being the most common. Despite advances in antisense oligonucleotide (ASO)-mediated exon skipping, therapeutic efficacy remains limited by suboptimal transcript availability and delivery barriers. We investigated whether histone deacetylase inhibitors (HDACis), particularly givinostat, can enhance the efficacy of an exon 51-targeting ASO in the clinically relevant mdx52 mouse model. For the first time, we identified a pronounced 5′–3′ DMD transcript imbalance associated with exon 52 deletion, contrasting with the classical mdx model and potentially compromising ASO effectiveness. In human DMD myoblasts carrying an exon 52 deletion, givinostat significantly improved exon skipping and dystrophin restoration. In vivo, givinostat and ASO co-treatment resulted in a modest but significant increase in dystrophin levels compared to ASO alone (1.3-fold), along with reduced muscle fibrosis, improved fiber morphology, and better extracellular matrix organization. Notably, treated mice showed improved muscle function, as evidenced by a significant reduction in force loss after eccentric contractions, a clinically meaningful outcome. Although the precise molecular mechanisms underlying these effects remain to be fully elucidated, these findings support the therapeutic potential of combining givinostat with ASOs to enhance dystrophin restoration and muscle preservation in DMD.
Xaysongkhame Phongsavanh, Cécile Gastaldi, Astrid Mottais et al.· Molecular Therapy: Nucleic A...· 0 citations
Background LMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. Results In this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. Conclusions Our findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.
M. Santafé, I. Hernández, D. Mazzeo et al.· bioRxiv· 0 citations
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disorder caused by mutations in the DMD gene, resulting in the absence of functional dystrophin. The loss of dystrophin disrupts the dystrophin-associated protein complex (DAPC), leading to progressive degeneration of skeletal and cardiac muscles. Clinically, DMD is characterized by proximal muscle weakness, respiratory insufficiency, and dilated cardiomyopathy. Here, we report the generation of three induced pluripotent stem cell (iPSC) lines reprogrammed from peripheral blood mononuclear cells (PBMCs) of patients with DMD. These patient-derived iPSC lines constitute a relevant human cellular model for studying disease-associated phenotypes and evaluating potential therapeutic strategies.
Hamel Mahiou, Benjamin Marande, L. Kassar et al.· Stem Cell Research· 0 citations