邦武 克彦

Antisense oligonucleotide (ASO)-based exon skipping is a representative splice modulating therapy effective for Duchenne muscular dystrophy (DMD), a condition arising from dystrophin deficiency. For a valuable platform of evaluating exon-skipping efficacy, we previously reported a highly effective approach to convert CD90-positive human urine-derived cells (UDCs) into myotubes through the transduction of the MYOD1 gene. We anticipate that highly differentiated myotubes enable their utilization in various human muscle cell studies and a more predictive screening approach for the preclinical identification of promising ASOs at an earlier stage of the drug discovery process. Here, we describe protocols and tips for isolating UDCs, differentiating CD90-positive UDCs into myotubes, and evaluating the correction of DMD mRNA and protein levels in the myotubes after exon skipping.

Antisense oligonucleotide (ASO)-based exon skipping is a splice-modulating therapy effective for Duchenne muscular dystrophy (DMD) caused by dystrophin deficiency. Here, we present a protocol for evaluating exon skipping efficacy in MYOD1-transduced human urine-derived cells (MYOD1-UDCs) from patients. We describe steps for isolating UDCs, selecting CD90-positive cells, inducing myogenic differentiation, and assessing the restoration of DMD mRNA and proteins after exon skipping. This platform enhances the predictability of ASO screening, promoting early-stage drug discovery and translational research in DMD. For complete details on the use and execution of this protocol, please refer to Komaki et al.1.

Duchenne muscular dystrophy (DMD) is a severe muscle disorder caused by mutations in the DMD gene, leading to dystrophin deficiency. Antisense oligonucleotide (ASO)-mediated exon skipping offers potential by partially restoring dystrophin, though current therapies remain mutation specific with limited efficacy. To overcome those limitations, we developed brogidirsen, a dual-targeting ASO composed of two directly connected 12-mer sequences targeting exon 44 using phosphorodiamidate morpholino oligomers. An open-label, dose-escalation, phase 1/2 trial assessed the safety, pharmacokinetics, and efficacy of brogidirsen in six ambulant patients with DMD amenable to exon 44 skipping. Following dose escalation, extended 24-week treatment with 40 mg/kg and 80 mg/kg yielded dose-dependent increases in dystrophin (16.63% and 24.47% of normal). Functional assessments indicated motor stabilization, and plasma proteomics revealed reductions in peptidyl arginine deiminase 2 (PADI2), titin (TTN), and myomesin 2 (MYOM2), highlighting potential biomarkers. Brogidirsen's efficacy was supported in vitro using urine-derived cells from patients with DMD. These promising results warrant a subsequent trial for DMD. This study was registered at ClinicalTrials.gov (NCT04129294).

Human urine-derived cells (UDCs) are primary cultured cells originating from the upper urinary tract and are known to be multipotent. We previously developed MYOD1-transduced UDCs (MYOD1-UDCs) as a model recapitulating the pathogenesis of Duchenne muscular dystrophy (DMD) caused by a lack of dystrophin. MYOD1-UDCs also allow evaluation of the efficacy of exon skipping with antisense oligonucleotides. However, despite the introduction of MYOD1, some MYOD1-UDCs failed to form myotubes, possibly because of heterogeneity among UDCs. Here, we carried out single-cell RNA-sequencing analyses and revealed that CD90/Thy-1 was highly expressed in a limited subpopulation of UDCs with high myogenic potency. Furthermore, CD90-positive MYOD1-UDCs, but not CD90-negative cells, could form myotubes expressing high levels of myosin heavy chain and dystrophin. Notably, overexpression of CD90 in CD90-negative MYOD1-UDCs did not enhance myogenic differentiation, whereas CD90 suppression in CD90-positive UDCs led to decreased myotube formation and decreased myosin heavy chain expression. CD90 may thus contribute to the fusion of single-nucleated MYOD1-UDCs into myotubes but is not crucial for promoting the expression of late muscle regulatory factors. Finally, we confirmed that CD90-positive MYOD1-UDCs derived from patients with DMD were a valuable tool for obtaining a highly reproducible and stable evaluation of exon skipping using antisense oligonucleotide.

Antisense oligonucleotide (ASO)-mediated exon skipping has become a valuable tool for investigating gene function and developing gene therapy. Machine-learning-based computational methods, such as eSkip-Finder, have been developed to predict the efficacy of ASOs via exon skipping. However, these methods are computationally demanding, and the accuracy of predictions remains suboptimal. In this study, we propose a new approach to reduce the computational burden and improve the prediction performance by using feature selection within machine-learning algorithms and ensemble-learning techniques. We evaluated our approach using a dataset of experimentally validated exon-skipping events, dividing it into training and testing sets. Our results demonstrate that using a three-way-voting approach with random forest, gradient boosting, and XGBoost can significantly reduce the computation time to under ten seconds while improving prediction performance, as measured by R2 for both 2'-O-methyl nucleotides (2OMe) and phosphorodiamidate morpholino oligomers (PMOs). Additionally, the feature importance ranking derived from our approach is in good agreement with previously published results. Our findings suggest that our approach has the potential to enhance the accuracy and efficiency of predicting ASO efficacy via exon skipping. It could also facilitate the development of novel therapeutic strategies. This study could contribute to the ongoing efforts to improve ASO design and optimize gene therapy approaches.