Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers. Here, we present an alternative gene therapy approach targeting reduced ASM secretion, the consequence of dysferlin deficit. We showed that the bulk endocytic ability is compromised in LGMD2B patient cells, which was addressed by extracellularly treating cells with ASM. Expression of secreted human ASM (hASM) using a liver-specific adeno-associated virus (AAV) vector restored membrane repair capacity of patient cells to healthy levels. A single in vivo dose of hASM-AAV in the LGMD2B mouse model restored myofiber repair capacity, enabling efficient recovery of myofibers from focal or lengthening contraction–induced injury. hASM-AAV treatment was safe, attenuated fibro-fatty muscle degeneration, increased myofiber size, and restored muscle strength, similar to dysferlin gene therapy. These findings elucidate the role of ASM in dysferlin-mediated plasma membrane repair and to our knowledge offer the first non–muscle-targeted gene therapy for LGMD2B.

中文翻译：

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分泌型酸性鞘磷脂酶作为肢带型肌营养不良症 2B 的潜在基因疗法


肌肉细胞的生存需要有效的肌膜修复，这一过程的缺陷会导致肌肉退化。肌膜蛋白 Dysferlin 的缺乏会通过减少酸性鞘磷脂酶 (ASM) 的分泌来损害肌膜修复，并导致肢带型肌营养不良症 2B (LGMD2B)。 Dysferlin 基因的大尺寸对旨在恢复骨骼肌纤维中 Dysferlin 表达的 LGMD2B 基因治疗工作提出了挑战。在这里，我们提出了一种替代基因治疗方法，目标是减少 ASM 分泌，这是 Dysferlin 缺陷的结果。我们发现 LGMD2B 患者细胞的大量内吞能力受到损害，通过用 ASM 对细胞进行细胞外处理可以解决这一问题。使用肝脏特异性腺相关病毒 (AAV) 载体表达分泌的人 ASM (hASM)，使患者细胞的膜修复能力恢复到健康水平。 LGMD2B 小鼠模型中的单剂量体内 hASM-AAV 恢复了肌纤维修复能力，使肌纤维能够从局部或延长收缩引起的损伤中有效恢复。 hASM-AAV 治疗是安全的，可以减轻纤维脂肪肌肉变性，增加肌纤维尺寸，并恢复肌肉强度，类似于 Dysferlin 基因治疗。这些发现阐明了 ASM 在 Dysferlin 介导的质膜修复中的作用，并且据我们所知，为 LGMD2B 提供了第一个非肌肉靶向基因疗法。