A NEW DYSTROPHIN DEFICIENT RAT MODEL MIRRORING EXON SKIPPING IN PATIENTS WITH DMD EXON 45 DELETIONS

Mutations in the dystrophin (DMD) gene can 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 frequently reported single-exon deletion in DMD patients worldwide. In this study, we generated a novel rat model with an exon 45 deletion using the CRISPR/Cas9 technology. The DmdΔ45 rat recapitulate key clinical and molecular features of DMD, including progressive skeletal muscle degeneration, impaired muscle and cardiac function, cognitive deficits, elevated circulating muscle damage biomarkers and an overall reduced lifespan. Transcriptomics analyses confirmed the deletion of exon 45 and revealed gene expression patterns consistent with dystrophin deficiency. In the skeletal muscle, RNA-seq profiles demonstrated a transition from early stress responses and regenerative activity at 6 months to chronic inflammation, fibrosis, and metabolic dysfunction by 12 months. Similarly, the cardiac transcriptomic shifted from an early inflammatory and stress-responsive state to one characterized by fibrotic remodelling and metabolic impairment. Despite these pathological features, the DmdΔ45 rats exhibited a milder phenotype than other DMD rat models. This attenuation may be attributed to spontaneous exon 44 skipping, which partially restores the reading frame and results in an age-dependent increase in revertant dystrophin-positive fibres. Further analysis indicated downregulation of spliceosome-related genes, suggesting a potential mechanism driving exon skipping in this model. In summary, the DmdΔ45 rat represents a valuable model for investigating both the molecular determinants of phenotypic variability and the endogenous mechanisms of exon skipping. These findings offer important insights for the development of personalized exon-skipping therapies, particularly for DMD patients with exon 45 deletions.抗肌萎缩蛋白（dystrophin, DMD）基因的突变可引发一系列肌肉萎缩类疾病，涵盖症状较轻的贝克型肌营养不良症（Becker muscular dystrophy, BMD）与更为严重的杜氏肌营养不良症（Duchenne muscular dystrophy, DMD）。外显子45缺失是全球范围内DMD患者中报道最为频繁的单外显子缺失类型。本研究借助CRISPR/Cas9技术构建了一种携带外显子45缺失的新型大鼠模型。该DmdΔ45大鼠能够重现DMD的关键临床与分子特征，包括进行性骨骼肌变性、肌肉与心脏功能受损、认知缺陷、循环肌损伤生物标志物水平升高以及整体寿命缩短。转录组学分析验证了外显子45的缺失，并揭示出与抗肌萎缩蛋白缺失相符的基因表达模式。在骨骼肌中，RNA测序（RNA-seq）谱显示基因表达从6月龄时的早期应激反应与再生活性，逐步过渡至12月龄时的慢性炎症、纤维化与代谢功能障碍。同样，心脏转录组也从早期炎症与应激应答状态，转变为以纤维化重塑和代谢损伤为特征的状态。尽管存在上述病理特征，DmdΔ45大鼠的表型相较于其他DMD大鼠模型更为温和。这种表型减弱现象可归因于自发的外显子44跳跃：该过程可部分恢复阅读框，并导致年龄依赖性的回复性抗肌萎缩蛋白阳性纤维数量增加。进一步分析显示，剪接体相关基因存在下调现象，这提示了驱动本模型中外显子跳跃的潜在机制。综上，DmdΔ45大鼠是研究表型异质性的分子决定因素以及外显子跳跃内源机制的宝贵模型。本研究结果为个性化外显子跳跃疗法的开发提供了重要见解，尤其针对携带外显子45缺失的DMD患者。