Current gene therapy for Duchenne muscular dystrophy (DMD) utilizes adeno-associated virus (AAV) to deliver micro-dystrophin (µDys), which does not provide full protection for striated muscles as it lacks many important functional domains of full-length (FL) dystrophin. Here we develop a triple vector system to deliver FL-dystrophin into skeletal and cardiac muscles. We split FL-dystrophin into three fragments linked to two orthogonal pairs of split intein, allowing efficient assembly of FL-dystrophin. The three fragments packaged in myotropic AAV (MyoAAV4A) restore FL-dystrophin expression in both skeletal and cardiac muscles in male mdx^4cv mice. Dystrophin-glycoprotein complex components are also restored at the sarcolemma of dystrophic muscles. MyoAAV4A-delivered FL-dystrophin significantly improves muscle histopathology, contractility, and overall strength comparable to µDys, but unlike µDys, it also restores defective cavin 4 localization and associated signaling in mdx^4cv heart. Therefore, our data support the feasibility of a mutation-independent FL-dystrophin gene therapy for DMD, warranting further clinical development.

目前针对杜氏肌营养不良症 (DMD) 的基因疗法利用腺相关病毒 (AAV) 传递微肌营养不良蛋白 (μDys)，但由于缺乏全长 (FL) 的许多重要功能域，因此无法为横纹肌提供全面保护肌营养不良蛋白。在这里，我们开发了一个三重载体系统，将 FL-肌营养不良蛋白输送到骨骼肌和心肌中。我们将 FL-肌营养不良蛋白分成三个片段，连接到两对正交的分裂内含肽，从而实现 FL-肌营养不良蛋白的有效组装。包装在肌向性 AAV (MyoAAV4A) 中的三个片段可恢复雄性mdx ^4cv小鼠骨骼肌和心肌中 FL-肌营养不良蛋白的表达。肌营养不良蛋白-糖蛋白复合物成分也在营养不良肌肉的肌膜处得到恢复。与 µDys 相比，MyoAAV4A 递送的 FL-肌营养不良蛋白显着改善了肌肉组织病理学、收缩性和整体强度，但与 µDys 不同的是，它还恢复了mdx ^4cv心脏中缺陷的 Cavin 4 定位和相关信号传导。因此，我们的数据支持不依赖突变的 FL-肌营养不良蛋白基因治疗 DMD 的可行性，值得进一步的临床开发。