Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Sci Adv. 2020 Feb 19;6(8):eaay6812. doi: 10.1126/sciadv.aay6812. eCollection 2020 Feb.
Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (). Previously, we applied CRISPR-Cas9-mediated "single-cut" genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9-mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.
杜氏肌营养不良症（DMD）是一种致命的神经肌肉疾病，由肌营养不良蛋白基因（）突变引起。此前，我们应用 CRISPR-Cas9 介导的“单切”基因组编辑技术，在 DMD 的动物模型中纠正了多种遗传突变。然而，高效的体内基因组编辑需要高剂量的腺相关病毒（AAV），这给临床应用带来了挑战。在这项研究中，我们将 Cas9 核酸酶包装在单链 AAV（ssAAV）中，并将 CRISPR 单指导 RNA 包装在自我互补 AAV（scAAV）中，然后将这种双 AAV 系统递送到 DMD 小鼠模型中。高效基因组编辑所需的 scAAV 剂量至少比 ssAAV 低 20 倍。接受系统治疗的小鼠表现出肌营养不良蛋白表达的恢复和肌肉收缩力的改善。这些发现表明，使用 scAAV 系统可以显著提高 CRISPR-Cas9 介导的基因组编辑效率。这代表着朝着 DMD 的基因组编辑治疗转化迈出了重要一步。
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