Center for Gene Therapy, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio, USA.
Sarepta Therapeutics, Inc., Cambridge, Massachusetts, USA.
Hum Gene Ther. 2021 Apr;32(7-8):390-404. doi: 10.1089/hum.2019.199. Epub 2021 Feb 18.
Limb-girdle muscular dystrophy type 2D/R3 (LGMD2D/R3) is a progressive muscular dystrophy that manifests with muscle weakness, respiratory abnormalities, and in rare cases cardiomyopathy. LGMD2D/R3 is caused by mutations in the SGCA gene resulting in loss of protein and concomitant loss of some or all components of the dystrophin-associated glycoprotein complex. The sgca-null () mouse recapitulates the clinical phenotype of patients with LGMD2D/R3, including dystrophic features such as muscle necrosis and fibrosis, elevated serum creatine kinase (CK), and reduction in the generation of absolute muscle force and locomotor activity. Thus, mice provide a relevant model to test the safety and efficacy of gene transfer. We designed a self-complementary AAVrh74 vector containing a codon-optimized full-length human SGCA (hSGCA) transgene driven by a muscle-specific promoter, shortened muscle creatine kinase (tMCK). In this report, we test the efficacy and safety of scAAVrh74.tMCK.hSGCA in mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 10, 3.0 × 10, and 6.0 × 10 vg total dose compared with vehicle-treatment and wild-type mice. In mice, treatment with scAAVrh74.tMCK.hSGCA resulted in robust expression of α-sarcoglycan protein at the sarcolemma membrane in skeletal muscle at all doses tested. In addition, scAAVrh74.tMCK.hSGCA was effective in improving the histopathology of limb and diaphragm muscle of mice, as indicated by reductions in fibrosis, central nucleation, and normalization of myofiber size. These molecular changes were concomitant with significant increases in specific force generation in the diaphragm and tibialis anterior muscle, protection against eccentric force loss, and reduction in serum CK. Locomotor activity was improved at all doses of vector-treated compared with vehicle-treated mice. Lastly, vector toxicity was not detected in a serum chemistry panel and by gross necropsy. Collectively, these findings provide support for a systemic delivery of scAAVrh74.tMCK.hSGCA in a clinical setting for the treatment of LGMD2D/R3.
2D/3R 型肢带型肌营养不良症（LGMD2D/R3）是一种进行性肌营养不良症，其特征为肌肉无力、呼吸异常，在极少数情况下还伴有心肌病。LGMD2D/R3 是由 SGCA 基因突变引起的，导致蛋白丢失以及伴随之一部分或全部的抗肌萎缩蛋白糖复合物成分丢失。sgca 敲除（）小鼠重现了 LGMD2D/R3 患者的临床表型，包括肌肉坏死和纤维化等营养不良特征、血清肌酸激酶（CK）升高以及绝对肌肉力量和运动活动生成减少。因此，小鼠提供了一个相关的模型来测试基因转移的安全性和疗效。我们设计了一种自我互补的 AAVrh74 载体，其中包含一个由肌肉特异性启动子驱动的、密码子优化的全长人 SGCA（hSGCA）转基因，缩短了肌肉肌酸激酶（tMCK）。在本报告中，我们使用剂量递增设计测试了 scAAVrh74.tMCK.hSGCA 在小鼠中的疗效和安全性，以评估与载体处理和野生型小鼠相比，单次全身注射 1.0×10、3.0×10 和 6.0×10 vg 总剂量的效果。在小鼠中，用 scAAVrh74.tMCK.hSGCA 治疗可导致α- 连接蛋白在所有测试剂量的骨骼肌肌细胞膜上的膜表达。此外，scAAVrh74.tMCK.hSGCA 有效改善了小鼠的肢体和膈肌肌肉的组织病理学，表现为纤维化、中央核化和肌纤维大小的正常化减少。这些分子变化伴随着膈肌和胫骨前肌特定力量生成的显著增加、对抗离心力损失的保护以及血清 CK 的减少。与载体处理的车辆处理的小鼠相比，运动活动在所有载体剂量下都得到了改善。最后，在血清化学小组和大体解剖学中未检测到载体毒性。总的来说，这些发现为在临床环境中进行全身性递送 scAAVrh74.tMCK.hSGCA 治疗 LGMD2D/R3 提供了支持。
