Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.

中文翻译：

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通过在 mdx 小鼠中使用 FORCE 结合的 TfR1 靶向递送反义寡核苷酸实现增强的外显子跳跃和延长的抗肌萎缩蛋白恢复

Duchenne 肌营养不良症 (DMD) 的当前疗法使用二氨基磷酸酯吗啉寡聚体 (PMO) 来诱导抗肌萎缩蛋白前体 mRNA 中的外显子跳跃，从而能够翻译缩短但具有功能的抗肌萎缩蛋白。PMO 向心肌和骨骼肌的递送不足阻碍了该策略。为了克服这些限制，我们开发了由抗原结合片段组成的 FORCETM 平台，该片段结合转铁蛋白受体 1，与寡核苷酸结合。我们证明单剂量的小鼠特异性 FORCE-M23D 偶联物可增强 mdx 小鼠中外显子跳跃 PMO (M23D) 的肌肉递送，实现剂量依赖性和稳健的外显子跳跃和持久的抗肌萎缩蛋白恢复。FORCE–M23D 诱导的抗肌萎缩蛋白表达在四头肌中达到野生型水平的 51%、72%、62%、90% 和 77% 的峰值，胫骨前肌、腓肠肌、膈肌和心脏，分别使用单次 30 mg/kg PMO 等效剂量。缩短的抗肌萎缩蛋白定位于肌膜，表明功能蛋白的表达。相反，单次 30 mg/kg 剂量的未结合 M23D 表现出较差的肌肉输送，导致边缘水平的外显子跳跃和抗肌萎缩蛋白表达。重要的是，与未结合的 M23D 给药相比，FORCE–M23D 治疗可改善功能结果。我们的结果表明，FORCE 偶联物是治疗 DMD 的潜在有效方法。单次 30 mg/kg 剂量的未结合 M23D 表现出较差的肌肉输送，导致边缘水平的外显子跳跃和抗肌萎缩蛋白表达。重要的是，与未结合的 M23D 给药相比，FORCE–M23D 治疗可改善功能结果。我们的结果表明，FORCE 偶联物是治疗 DMD 的潜在有效方法。单次 30 mg/kg 剂量的未结合 M23D 表现出较差的肌肉输送，导致边缘水平的外显子跳跃和抗肌萎缩蛋白表达。重要的是，与未结合的 M23D 给药相比，FORCE–M23D 治疗可改善功能结果。我们的结果表明，FORCE 偶联物是治疗 DMD 的潜在有效方法。