Limb-girdle muscular dystrophy type 2A (LGMD2A or LGMDR1) is a neuromuscular disorder caused by mutations in the calpain 3 gene (CAPN3). Previous experiments using adeno-associated viral (AAV) vector–mediated calpain 3 gene transfer in mice indicated cardiac toxicity associated with the ectopic expression of the calpain 3 transgene. Here, we performed a preliminary dose study in a severe double-knockout mouse model deficient in calpain 3 and dysferlin. We evaluated safety and biodistribution of AAV9-desmin-hCAPN3 vector administration to nonhuman primates (NHPs) with a dose of 3 × 10¹³ viral genomes/kg. Vector administration did not lead to observable adverse effects or to detectable toxicity in NHP. Of note, the transgene expression did not produce any abnormal changes in cardiac morphology or function of injected animals while reaching therapeutic expression in skeletal muscle. Additional investigation on the underlying causes of cardiac toxicity observed after gene transfer in mice and the role of titin in this phenomenon suggest species-specific titin splicing. Mice have a reduced capacity for buffering calpain 3 activity compared to NHPs and humans. Our studies highlight a complex interplay between calpain 3 and titin binding sites and demonstrate an effective and safe profile for systemic calpain 3 vector delivery in NHP, providing critical support for the clinical potential of calpain 3 gene therapy in humans.

肢带型肌营养不良症2A型（LGMD2A或LGMDR1）是钙蛋白酶3基因（突变引起的神经肌肉病症CAPN3）。先前使用腺相关病毒（AAV）载体介导的calpain 3基因转移的实验表明，心脏毒性与calpain 3转基因的异位表达有关。在这里，我们在钙蛋白酶3和dysferlin缺乏的严重双敲除小鼠模型中进行了初步剂量研究。我们评估了AAV9-desmin-hCAPN3载体对3×10 ¹³剂量的非人类灵长类动物（NHP）的安全性和生物分布。病毒基因组/千克。在NHP中，载体给药未导致可观察到的不良反应或可检测到的毒性。值得注意的是，转基因表达在骨骼肌中达到治疗性表达时，不会在注射的动物的心脏形态或功能上产生任何异常变化。对小鼠基因转移后观察到的心脏毒性的根本原因进行的进一步研究以及titin在这种现象中的作用表明，存在种特异性的titin剪接。与NHP和人类相比，小鼠的钙蛋白酶3活性缓冲能力降低。我们的研究突出了钙蛋白酶3和替丁结合位点之间的复杂相互作用，并证明了在NHP中系统性钙蛋白酶3载体递送的有效和安全性，为钙蛋白酶3基因治疗人类的临床潜力提供了关键支持。