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 (sgca^−/−) 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, sgca^−/− 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 sgca^−/− mice using a dose-escalation design to evaluate a single systemic injection of 1.0 × 10¹², 3.0 × 10¹², and 6.0 × 10¹² vg total dose compared with vehicle-treatment and wild-type mice. In sgca^−/− 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 sgca^−/− 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 sgca^−/− 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.

中文翻译：

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腺相关α-肌聚糖基因转移治疗肢带型肌营养不良的临床前全身给药

2D/R3 型肢带型肌营养不良症 (LGMD2D/R3) 是一种进行性肌营养不良症，表现为肌肉无力、呼吸异常，在极少数情况下会出现心肌病。LGMD2D/R3 是由 SGCA 基因突变引起的，导致蛋白质丢失，并伴随肌营养不良蛋白相关糖蛋白复合物的部分或全部成分丢失。sgca-null ( sgca ^-/- ) 小鼠概括了 LGMD2D/R3 患者的临床表型，包括营养不良特征，如肌肉坏死和纤维化、血清肌酸激酶 (CK) 升高以及绝对肌肉力量的产生减少和运动活动。因此，sgca ^-/-小鼠提供了一个相关模型来测试基因转移的安全性和有效性。我们设计了一个自我互补的 AAVrh74 载体，该载体包含由肌肉特异性启动子缩短的肌肉肌酸激酶 (tMCK) 驱动的密码子优化的全长人类 SGCA (hSGCA) 转基因。在本报告中，我们使用剂量递增设计测试了 scAAVrh74.tMCK.hSGCA 在sgca ^-/-小鼠中的有效性和安全性，以评估单次全身注射 1.0 × 10 ¹²、3.0 × 10 ¹²和 6.0 × 10 ¹² vg 总剂量与载体治疗和野生型小鼠相比。在sgca ^-/-小鼠，用 scAAVrh74.tMCK.hSGCA 处理导致在所有测试剂量下骨骼肌肌膜上 α-肌聚糖蛋白的强烈表达。此外，scAAVrh74.tMCK.hSGCA 可有效改善sgca ^-/-小鼠四肢和膈肌的组织病理学，如纤维化减少、中心成核和肌纤维尺寸正常化所示。这些分子变化伴随着膈肌和胫骨前肌比力产生的显着增加、防止离心力损失的保护以及血清 CK 的降低。与载体处理的sgca相比，所有剂量的载体处理的运动活性均得到改善^-/-老鼠。最后，在血清化学小组和大体尸检中未检测到载体毒性。总的来说，这些发现为在治疗 LGMD2D/R3 的临床环境中全身递送 scAAVrh74.tMCK.hSGCA 提供了支持。