Genethon (France)

Pathogenic variants in the dystrophin (DMD) gene cause muscle-wasting disorders ranging from the milder Becker muscular dystrophy (BMD) to the more severe Duchenne muscular dystrophy (DMD). Exon 45 deletion is the most-frequent single-exon deletion in patients diagnosed with DMD. Here, we generated a novel rat model with an exon 45 deletion using CRISPR/Cas9. The DmdΔ45 rat recapitulate key features of DMD, including progressive skeletal muscle degeneration, impaired muscle and cardiac function, and cognitive deficits. Transcriptomics analyses revealed gene expression patterns consistent with dystrophin deficiency. In skeletal muscle, we observed a transition from early stress responses and regeneration to chronic inflammation, fibrosis and metabolic dysfunction. Cardiac profiles similarly progressed from early inflammatory responses to fibrotic remodelling and metabolic impairment. Notably, DmdΔ45 rats displayed a milder phenotype than other DMD rat models. This attenuation is likely due to spontaneous exon skipping, particularly of exon 44, which partially restores the reading frame and increases revertant dystrophin-positive fibres with age. Downregulation of spliceosome-related genes suggests a potential mechanism for this exon skipping. Overall, this model provides valuable insights into phenotypic variability and therapeutic exon-skipping strategies.

Abstract Gene therapy has emerged as a promising curative treatment for β-hemoglobinopathies, the most common genetic disorders worldwide. However, current approved approaches still have some limitations in terms of safety and efficacy. Here, we used highly processive adenine base editors (ABE) variants to precisely correct some of the most prevalent and severe β-thalassemia-causing mutations in the β-globin gene. Efficient editing of hematopoietic stem/progenitor cells (HSPCs) led to potent β-globin expression in their erythroid progeny and persistent correction of both β-thalassemia and sickle cell-β-thalassemia phenotypes. Safety of this strategy was confirmed in HSPCs in vitro and in vivo by the absence of gene dysregulation or any meaningful impact on the DNA mutational burden, the RNA deamination level, the β-globin gene locus integrity and the clonality of the HSPC graft. Overall, base editing-mediated gene correction is a safe and effective strategy for treating β-hemoglobinopathies. One sentence summary Preclinical safety and efficacy studies of a new gene therapy approach for patients with severe β-hemoglobinopathies.

Genome editing technologies, particularly CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), have broadened the possibilities of genetic research and molecular biology by enabling precise modifications of the genome, offering novel therapeutic potential for various disorders. Herein, we present an overview of traditional genome editing techniques and delve deeper into the CRISPR toolbox, with particular attention given to epigenetic and transcriptional regulation. In the context of the intervertebral disc (IVD), CRISPR offers an unprecedented approach to address the mechanisms underlying tissue degeneration, advancing the development of revolutionary therapies for Low Back Pain (LBP). As so, we showcase how to leverage CRISPR systems for IVD. This cutting-edge technology has been successfully used to improve our understanding of IVD biology through functional studies and disease modeling. Most relevant research prioritizes new targets associated with the extracellular matrix (ECM), pain sensing or inflammatory pathways. Promising CRISPR applications encompass IVD regeneration by recapitulation of a regenerative environment or by targeting important degenerative catalysts. In the future, priority should be given to fetal gene reactivation, multiple healthy gene expression enhancement and disease-associated polymorphisms' correction. Despite several challenges such as effective delivery, off-target effects, as well as ethical and safety concerns, exciting clinical trials are anticipated in the years to come, providing more effective and long-lasting solutions for IVD degeneration.

Limb-girdle muscular dystrophy R2 (LGMD R2) is a rare genetic disorder characterised by progressive weakness and wasting of proximal muscles. LGMD R2 is caused by the loss of function of dysferlin, a transmembrane protein crucial for plasma membrane repair in skeletal muscles. This study aimed to identify drugs that could improve the localisation and restore the function of an aggregated mutant form of dysferlin (DYSFL1341P). We developed an in vitro high-throughput assay to monitor the expression and reallocation of aggregated mutant dysferlin (DYSFL1341P) in immortalised myoblasts. After screening 2239 clinically approved drugs and bioactive compounds, the ability of the more promising candidates to improve cell survival following hypo-osmotic shock was assessed. Their protective effects were evaluated on immortalised myoblasts carrying other dysferlin mutations and on dysferlin-deficient muscle fibres from Bla/J mice. We identified two compounds, saracatinib and bazedoxifene, that increase dysferlin content in cells carrying the DYSFL1341P mutation. Both drugs improved cell survival and plasma membrane resistance following osmotic shock. Whereas saracatinib acts specifically on misfolded L1341P dysferlin, bazedoxifene shows an additional protective effect on dysferlin KO immortalised myoblasts and mice muscle fibres. Further analysis revealed that bazedoxifene induces autophagy flux, which may enhance the survival of LGMD R2 myofibres. Our drug screening identified saracatinib and bazedoxifene as potential treatments for LGMD R2, especially for patients with the L1341P mutation. The widespread protective effect of bazedoxifene reveals a new avenue toward genotype-independent treatment of LGMD R2 patients.

Wolman disease (WD) is a severe lysosomal storage disorder characterized by fatal lipid accumulation caused by the deficiency of a lipid metabolic enzyme, Lysosomal Acid Lipase (LAL), involved in the lysosomal hydrolysis of cholesterols and triglycerides. Due to the imbalance of lipids homeostasis, WD patients suffer from severe hepatosplenomegaly, hepatic failure and adrenal calcification resulting in a premature infant death within the first year of age. In this work, we explored multiple imaging analyses to fully characterize the phenotype of LAL deficient cells. In particular, we stained WD patients' fibroblasts for intracellular lipid droplets (LD) and lysosomes and we analysed staining intensity and granularity as well as an increased number of LD and lysosomes using fluorescence wide field microscopy, confocal microscopy, conventional and image flow cytometry. Noteworthy, we showed that lipid homeostasis was restored upon delivery of a functional LAL transgene. Finally, since fibroblasts cannot be used as routine clinical test as they are difficult to collect from WD patients, we confirmed our observations in LAL deficient human blood cell lines and in peripheral blood mononuclear cells (PBMC) from LAL deficient (LAL-D) mouse model, as a proxy for easily accessible WD PBMC. Overall, we expect that this novel imaging analysis pipeline will help to diagnose WD, follow its progression and evaluate the success of enzyme replacement therapy or gene correction strategies for WD as well as other lysosomal storage disorders.

Duchenne Muscular Dystrophy (DMD) is a pediatric disorder characterized by progressive muscle degeneration and premature death, and has no current cure. The current, most promising therapeutic avenue is based on gene replacement mediated by adeno-associated viruses (AAVs) using a shortened, but still functional, version of dystrophin, known as micro-dystrophin (µDys), to fit AAV capacity. The limited improvements observed in clinical trials suggest a sub-optimal performance of µDys in the human context that could be due to the lack of key domains in the protein. Therefore, expressing larger dystrophin proteins may be necessary for a more complete correction of the disease phenotype. In this study, we developed three novel midi-dystrophin constructs using a dual-AAV approach, leveraging split-intein-based protein trans-splicing. The midi-dystrophins include additional domains compared to µDys, such as the central cytoskeleton-binding domain, nNOS and Par1b interacting domains, and a complete C-terminal region. Given the limited capacity of each AAV vector, we strategically partially reduced hinge regions while ensuring that the structural stability of the protein remains intact. We predicted the interactions between the two halves of the split midi-Dys proteins thanks to the deep learning algorithm AphaFold3. We observed strong associations between the N- and C-termini in midi-Dys 1 and 2, while a weaker interaction in midi-Dys 3 was revealed. Our subsequent experiments confirmed the efficient protein trans-splicing both in vitro and in vivo in DBA2/mdx mice of the midi-Dys 1 and 2 and not in midi-Dys 3 as expected from the structural prediction. Additionally, we demonstrated that midi-Dys 1 and 2 exhibit significant therapeutic efficacy in DBA2/mdx mice, highlighting their potential as therapeutic agents for DMD. Overall, these findings highlight the potential of deep learning-based structural modeling for the generation of intein-based dystrophin versions and pose the basis for further investigation of these new midi-dystrophins versions for clinical studies.

Variants in MYH7 cause cardiomyopathies as well as myosin storage myopathy and Laing early-onset distal myopathy (MPD1). MPD1 is characterized by muscle weakness and atrophy usually beginning in the lower legs. Here, we generated iPSC lines from lymphoblastoid cells of three unrelated individuals heterozygous for the most common MPD1-causing variant; p.Lys1617del. iPSC lines showed typical morphology, expressed pluripotency markers, demonstrated trilineage differentiation potential, and had a normal karyotype. These lines represent the first iPSCs derived from MPD1 patients and complement existing MPD1 animal models. They can provide in vitro platforms to better understand and model MPD1 pathomechanisms and test therapies.