Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri, USA.
National Center for Advancing Translational Sciences, NIH, Rockville, Maryland, USA.
JCI Insight. 2018 Dec 6;3(23):124297. doi: 10.1172/jci.insight.124297.
Adeno-associated virus-mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.
腺相关病毒介导的（AAV 介导的）CRISPR 编辑是治疗遗传性疾病的革命性方法。为了治疗这些疾病，需要持续的、通常是终身的突变校正。不幸的是，这从未在 AAV CRISPR 治疗中得到证明。我们在杜氏肌营养不良症（DMD）的 mdx 模型中解决了这个问题。DMD 是由肌营养不良蛋白基因突变引起的。肌营养不良蛋白的缺乏导致运动丧失和心肌病。我们通过静脉内治疗 6 周大的小鼠，并在 18 个月时评估疾病的恢复情况。令人惊讶的是，骨骼肌中的正常肌营养不良蛋白得到了恢复。心肌中的肌营养不良蛋白也得到了恢复，但组织学和血液动力学没有得到改善。为了确定潜在的机制，我们评估了 CRISPR 编辑机制的组成部分。有趣的是，我们发现向导 RNA（gRNA）载体的不成比例耗竭。为了测试这是否是导致不良结果的原因，我们增加了 gRNA 载体的剂量并重复了该研究。这一策略显著增加了 18 个月时所有横纹肌中的肌营养不良蛋白恢复，并减少了纤维化。重要的是，骨骼肌功能和心脏血液动力学得到了显著增强。有趣的是，我们在肌肉内注射后没有看到 gRNA 载体的选择性耗竭。我们的结果表明，gRNA 载体的丢失是全身 AAV CRISPR 治疗的一个独特障碍。通过优化载体剂量可以克服这一障碍。
