In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.
In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.
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DOI：
10.1161/circresaha.117.310996
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发表时间：
2017-09-29
影响因子：
20.1
通讯作者：
Han R
中科院分区：
文献类型：
--
作者：
El Refaey M;Xu L;Gao Y;Canan BD;Adesanya TMA;Warner SC;Akagi K;Symer DE;Mohler PJ;Ma J;Janssen PML;Han R
Duchenne肌营养不良症(DMD)是一种严重的遗传性肌营养不良症，由dystrophin基因阅读框突变扰乱其蛋白表达而引起。营养不良性心肌病是DMD患者死亡的主要原因，目前还没有有效的治疗方法来阻止其进展。基因组编辑技术的最新进展为恢复dystrophin蛋白的表达提供了一种很有前途的治疗方法。然而，这种方法对DMD心功能的影响还有待评估。因此，我们评估了在单次系统注射重组腺相关病毒后，CRISPR(集群规则间隔短回文重复序列)介导的基因组编辑对mdx/utr+/−小鼠dystrophin表达和心功能的治疗效果。目的：研究CRISPR介导的基因组编辑对营养不良小鼠心脏肌营养不良蛋白表达和功能的有效性和生理学影响。在这里，我们将SaCas9/gRNA包装成AAV载体，并系统地将它们传递给mdx/utr+/−新生儿。我们发现CRIPSR介导的基因组编辑有效地切除了营养不良小鼠突变的外显子23，免疫荧光数据支持营养不良心肌中dystrophin蛋白表达恢复到接近40%的水平。此外，在肌营养不良蛋白缺乏的心脏中，心肌纤维的结构有了显著的恢复，纤维化程度也有所减轻。与未经治疗的对照组相比，CRISPR编辑的心肌也恢复了心脏乳头肌的收缩能力。此外，我们的定向深度测序结果证实了我们的AAV-CRISPR-CAS9策略在删除~23kb的基因组序列方面非常有效。这项研究为使用基于CRISPR的基因组编辑作为一种潜在的治疗方法在结构和功能上恢复营养不良心肌病提供了证据。
Duchenne muscular dystrophy (DMD) is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in DMD patients and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on DMD cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/− mice after a single systemic delivery of recombinant adeno-associated virus (AAV). To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. Here we packaged SaCas9/gRNA constructs into an AAV vector and systemically delivered them to mdx/Utr+/− neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared to untreated controls. Furthermore, our targeted deep sequencing results confirmed that our AAV-CRISPR-Cas9 strategy was very efficient in deleting the ~23 kb of intervening genomic sequences. This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.