Project 1

项目总结/摘要 杜氏肌营养不良症（DMD）是一种遗传性X连锁疾病，由基因突变引起， 编码肌营养不良蛋白，肌纤维完整性所需的蛋白质。抗肌萎缩蛋白基因是最大的 基因组由79个外显子组成。尽管有成千上万的个体DMD突变， 在人类中，这些突变集中在肌营养不良蛋白基因的热点区域。DMD 影响约1/5，000的男孩，其特征是进行性严重肌无力， 寿命缩短尽管通过各种方法（包括 成肌细胞转移，抗肌萎缩蛋白的病毒递送，和阿托伐他汀介导的外显子跳跃，仍然没有 治愈这种疾病。我们的方法是使用CRISPR/Cas9基因组编辑来永久纠正DMD， 在体内跳过或重构出生后肌肉组织中的突变型肌营养不良蛋白外显子。我们把这个策略 我的编辑这种基因组编辑方法消除了导致疾病的基因突变， 允许永久性地矫正肌肉结构和功能。我们提供CRISPR/Cas9组件 使用腺相关病毒-9（AAV9）递送系统，该系统已经显示出提供稳健的 在骨骼肌、心脏和脑中表达，这是DMD患者中受影响的主要组织。到目前为止， 成功纠正了几种DMD动物模型中的肌营养不良蛋白基因突变， 肌营养不良蛋白基因的热点区域。在上一个融资期，我们产生了几个其他DMD 动物模型覆盖了剩余的人类热点区域，并建议使用 CRISPR/Cas基因组编辑。尽管我们已经利用CRISPR/Cas基因组编辑取得了很大进展， 尽管我们的基因编辑疗法已经被证明是正确的DMD，但要将这种基因编辑疗法转化为临床，还有更多的工作要做。的 CRISPR/Cas9组件的递送效率需要优化， 校正后抗肌萎缩蛋白表达的持久性。此外，由于肌肉纤维有数百个细胞核， 我们需要了解CRISPR/Cas9基因组编辑在个体核水平上的发生。的 该项目的长期目标仍然是优化和适应CRISPR/Cas9介导的基因组编辑， 并最终利用这种方法来纠正人类的DMD突变。这个项目 继续代表着临床医生和基础科学家之间的密切合作， 一种新的治疗策略来永久治愈DMD。

Project Summary/Abstract Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. The dystrophin gene is one of the largest human genes and consists of 79 exons. Although there are thousands of individual DMD mutations that have been identified in humans, these mutations are concentrated in hot spot regions of the dystrophin gene. DMD affects approximately 1 in 5,000 boys and is characterized by progressive severe muscle weakness and a shortened lifespan. Despite intense efforts to find cures for DMD through a variety of approaches, including myoblast transfer, viral delivery of dystrophin, and oligonucleotide-mediated exon skipping, there remains no cure for this disease. Our approach is to use CRISPR/Cas9 genomic editing to permanently correct DMD by skipping or reframing the mutant dystrophin exons in postnatal muscle tissue in vivo. We refer to this strategy as Myoediting. This genome editing approach removes the genetic mutation responsible for the disease, allowing for permanent correction of muscle structure and function. We deliver the CRISPR/Cas9 components using an adeno-associated virus-9 (AAV9) delivery system which has been shown to provide robust expression in skeletal muscle, heart and brain, the major tissues affected in DMD patients. To date, we have successfully corrected the dystrophin gene mutation in several DMD animal models having mutations in key hot spot regions of the dystrophin gene. In the previous funding period, we generated several other DMD animals models covering the remaining human hot spot regions and propose to correct these mutations using CRISPR/Cas genomic editing. Although we have made much progress using CRISPR/Cas genomic editing to correct DMD, there remains more work to be done to translate this gene editing therapy to the clinic. The efficiency of delivering the CRISPR/Cas9 components needs to be optimized and questions remain as to the durability of dystrophin expression after correction. Furthermore, since muscle fibers have hundreds of nuclei, we need to understand the occurrence of CRISPR/Cas9 genomic editing at the individual nuclear level. The long-term goal of this project remains to optimize and adapt CRISPR/Cas9-mediated genome editing to postnatal muscle and ultimately to leverage this approach to correct DMD mutations in humans. This project continues to represent a close collaboration between clinicians and basic scientists sharing the common goal of advancing a new therapeutic strategy to permanently cure DMD.