Project 1

项目摘要/摘要 Duchenne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，这是一种肌肉纤维完整性所必需的蛋白质。营养不良蛋白基因是最大的基因之一 人类基因，由79个外显子组成。尽管有数以千计的个体DMD突变 在人类中已被发现，这些突变集中在Dstrophin基因的热点区域。DMD 大约每5,000名男孩中就有1人受到影响，其特征是进行性严重的肌肉无力和 寿命缩短。尽管通过各种方法努力寻找治疗DMD的方法，包括 成肌细胞转移，Dystrophin病毒传递，以及寡核苷酸介导的外显子跳过，仍然没有 治愈这种疾病。我们的方法是使用CRISPR/Cas9基因组编辑通过以下方式永久纠正DMD 在活体内跳过或重构出生后肌肉组织中突变的肌营养不良蛋白外显子。我们指的是这一战略 作为MyoEditing。这种基因组编辑方法去除了导致这种疾病的基因突变， 允许永久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，已被证明提供了强大的 在骨骼肌、心脏和脑中表达，这是DMD患者受影响的主要组织。到目前为止，我们有 在几个关键基因突变的DMD动物模型中成功纠正了Dstrophin基因突变 Dystrophin基因的热点区域。在上一个资助期，我们生成了其他几个DMD 覆盖剩余人类热点区域的动物模型，并建议使用 CRISPR/Cas基因组编辑。尽管我们使用CRISPR/CAS基因组编辑技术已经取得了很大的进展 纠正DMD，要将这种基因编辑疗法转化为临床，还有更多的工作要做。这个 交付CRISPR/CAS9组件的效率需要优化，仍然存在以下问题 矫正后抗肌营养不良蛋白表达的持久性。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。这个 该项目的长期目标仍然是优化和调整CRISPR/Cas9介导的基因组编辑以 并最终利用这一方法来纠正人类的DMD突变。这个项目 继续代表临床医生和基础科学家之间的密切合作，共享共同的目标 提出了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.