Project 1

项目1

• 批准号: 10473541
• 负责人: ERIC N Olson
• 金额: $ 53.29万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10473541
• 关键词: Affect; Aging; Animal Model; Animal Muscular Dystrophy; Applications Grants; Brain; CRISPR/Cas technology; Canis familiaris; Cell Nucleus; Cell model; Cessation of life; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; DNA Sequence Alteration; Dependovirus; Disease; Dose; Duchenne muscular dystrophy; Dystrophin; Exercise; Exons; Funding; Gene Expression Profile; Gene Mutation; Genes; Genome; Genomic approach; Genomics; Goals; Heart; Hot Spot; Human; Individual; Inherited; Injury; Link; Longevity; Luciferases; Mediating; Mus; Muscle; Muscle Fibers; Muscle Weakness; Muscle function; Muscle satellite cell; Muscular Dystrophies; Mutation; Myoblasts; Myocardium; Nuclear; Oligonucleotides; Patients; Proteins; Reading Frames; Reporter; Scientist; Skeletal Muscle; System; Technology; Therapeutic; Time; Tissues; Translating; United States National Institutes of Health; Viral; Work; boys; disease-causing mutation; exon skipping; gene correction; genome editing; in vivo; in vivo monitoring; mouse model; muscular dystrophy mouse model; muscular structure; mutant; non-invasive monitor; novel therapeutic intervention; postnatal; premature; prevent; response; skeletal muscle wasting; stem cells; therapeutic genome editing; transcriptome sequencing

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.

项目概要/摘要 杜氏肌营养不良症 (DMD) 是一种由基因突变引起的遗传性 X 连锁疾病 编码肌营养不良蛋白，一种肌纤维完整性所需的蛋白质。抗肌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。尽管有数千种 DMD 突变 在人类中发现，这些突变集中在肌营养不良蛋白基因的热点区域。 DMD 大约五千分之一的男孩受到影响，其特征是进行性严重肌肉无力和 寿命缩短。尽管人们通过各种方法努力寻找 DMD 的治疗方法，包括 成肌细胞转移、抗肌营养不良蛋白的病毒传递和寡核苷酸介导的外显子跳跃，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 跳过或重构体内出生后肌肉组织中的突变肌营养不良蛋白外显子。我们参考这个策略 作为 Myoediting。这种基因组编辑方法消除了导致疾病的基因突变， 允许永久纠正肌肉结构和功能。我们提供 CRISPR/Cas9 组件 使用腺相关病毒 9 (AAV9) 递送系统，该系统已被证明可以提供强大的 在 DMD 患者受影响的主要组织骨骼肌、心脏和大脑中表达。迄今为止，我们已经 成功纠正了几种关键突变的DMD动物模型中的肌营养不良蛋白基因突变 抗肌萎缩蛋白基因的热点区域。在之前的资助期间，我们产生了其他几个 DMD 动物模型覆盖了剩余的人类热点区域，并建议使用以下方法来纠正这些突变 CRISPR/Cas 基因组编辑。尽管我们在使用 CRISPR/Cas 基因组编辑技术方面取得了很大进展 正确的DMD，将这种基因编辑疗法转化为临床还有更多的工作要做。这 CRISPR/Cas9 组件的传递效率需要优化，但仍然存在问题 校正后肌营养不良蛋白表达的持久性。此外，由于肌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 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 产后肌肉，并最终利用这种方法来纠正人类 DMD 突变。这个项目 继续代表着拥有共同目标的临床医生和基础科学家之间的密切合作 提出一种永久治愈 DMD 的新治疗策略。