Muscle-Specific CRISPR/Cas9 Exon Skipping for Duchenne Muscular Dystrophy Therapeutics

肌肉特异性 CRISPR/Cas9 外显子跳跃用于杜氏肌营养不良疗法

• 批准号: 10679199
• 负责人: Carolyn Kraus
• 金额: $ 3.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679199
• 关键词: 3' Untranslated Regions; 5 year old; Ablation; Address; Adrenal Cortex Hormones; Affect; Antisense Oligonucleotides; Bioinformatics; CRISPR/Cas technology; Cardiac Myocytes; Cell Line; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Consensus Sequence; Dependovirus; Development; Duchenne muscular dystrophy; Dystrophin; Epithelial Cells; Exons; FDA approved; Gene Mutation; Genes; Genetic; Genus staphylococcus; Guide RNA; Health; Heart; Infusion procedures; Inherited; Liver; Lung; Measures; Mediating; Messenger RNA; MicroRNAs; Modeling; Mouse Cell Line; Mus; Muscle; Muscle Cells; Muscle Weakness; Muscle function; Muscular Atrophy; Muscular Dystrophies; Mutation; Myoblasts; Myopathy; Neisseria meningitidis; Orthologous Gene; Other Genetics; Patients; Pharmaceutical Preparations; Plasmids; Proteins; RNA Splicing; Reading Frames; Repression; Risk; Safety; Site; Specificity; Symptoms; System; Testing; Therapeutic; Tissues; Wild Type Mouse; Work; adeno-associated viral vector; design; exon skipping; flexibility; improved; in vivo; inhibitor; mouse model; muscle strength; muscular dystrophy mouse model; nuclease; postnatal; promoter; respiratory; side effect; tissue tropism; tool; vector

PROJECT SUMMARY Duchenne muscular dystrophy (DMD)—a fatal inherited muscular dystrophy—is caused by loss of Dystrophin, a protein that maintains muscle integrity. Corticosteroids slow DMD progression but cause side effects. Addressing the root cause of DMD may improve patient health without needing corticosteroids. Many DMD- causing mutations disrupt the dystrophin mRNA reading frame, resulting in non-functional protein. Strategies that skip the out-of-frame exon to restore the reading frame and produce semi-functional protein for improved muscle function could correct 64% of DMD mutations. FDA-approved antisense oligonucleotide drugs can skip select exons in dystrophin mRNA, but require lifelong infusions and only work in a small group of patients. Using CRISPR to edit dystrophin would require just one treatment. CRISPR-mediated ablation of splice sites to cause exon skipping can increase Dystrophin in DMD models. Yet, editing in unintended tissues is a safety concern for Cas9 therapies. An ideal platform for DMD would restrict editing to muscle tissue to maximize therapeutic benefit. Efforts to achieve tissue-specific editing often rely on delivery via adeno-associated viruses (AAVs) with tissue tropism; yet, it is rarely absolute. Tissue-specific editing was recently achieved using tissue-specific miRNAs to regulate expression of Cas9 inhibitors [anti-CRISPR (Acr) proteins] via miRNA target sites (TS) in the 3’ UTR of Acr mRNA. When the platform is systemically delivered to mice via AAV, Acr-TS targeted by liver-specific miRNA allows editing only in the liver. Unlike tissue-specific promoters, this Acr-TS strategy could be adapted to one or multiple muscle tissues affected in DMD, as long as muscle-specific (myo)-miRNA can repress an Acr. With support from Erik Sontheimer (CRISPR, Acr), Eric Olson (DMD), Wen Xue (in vivo CRISPR delivery), Phillip Zamore (miRNA), Guangping Gao (AAV), and Zhiping Weng (bioinformatics), this proposal seeks to develop a muscle-specific editing platform to treat DMD. The myo-miRNA, miR-1, can repress an Acr in muscle cell lines to achieve muscle-specific editing. To fine-tune specificity of editing in muscle tissues for DMD, Aim 1 will test the ability of myo-miRs varying in abundance and muscle-type specificity to repress Acr and drive muscle-specific editing in mouse cell lines. The myo-miR construct supporting highest muscle-specific editing will be delivered to a DMD mouse model, and in vivo muscle function as well as dystrophin exon skipping, Dystrophin protein, and miRNA level in muscle tissues and liver will be measured. Aim 2 will test the compatibility of additional Cas9 orthologs in the Acr-TS system to enable targeting of more sequences, and develop a single AAV delivery system for improved safety. An Acr inhibiting the Cas9s to be tested has been identified. The ability of miR-1 to repress this Acr and drive muscle specific editing by each Cas9 will be tested in cells. A single vector encoding the Acr- TS system will be designed and packaged into AAV, and muscle-specific editing will be compared to a dual AAV system in mice. This work will develop a flexible, safe, muscle-specific CRISPR platform with the potential to be used for any combination of muscle tissues to treat patients with DMD, or other genetic muscle disorders.

项目概要 杜氏肌营养不良症 (DMD)——一种致命的遗传性肌营养不良症——是由肌营养不良蛋白缺失引起的， 一种维持肌肉完整性的蛋白质，可以减缓 DMD 的进展，但会引起副作用。 解决 DMD 的根本原因可以改善患者的健康，而无需使用皮质类固醇。 突变破坏肌营养不良蛋白 mRNA 阅读框，导致蛋白质无功能。 跳过框外外显子以恢复阅读框并产生半功能蛋白以改进 肌肉功能可以纠正 64% 的 DMD 突变，FDA 批准的反义寡核苷酸药物可以跳过。 选择肌营养不良蛋白 mRNA 中的外显子，但需要终生输注，并且仅对一小部分患者有效。 CRISPR 编辑肌营养不良蛋白只需要一种 CRISPR 介导的剪接位点消融即可引起。 外显子跳跃可以增加 DMD 模型中的肌营养不良蛋白，然而，在非预期组织中进行编辑是一个安全问题。 Cas9 疗法。DMD 的理想平台将限制对肌肉组织的编辑，以最大限度地提高治疗效果。 实现组织特异性编辑的努力通常依赖于通过腺相关病毒（AAV）与组织一起传递 向性；然而，最近使用组织特异性 miRNA 实现了组织特异性编辑。 通过 3' UTR 中的 miRNA 靶位点 (TS) 调节 Cas9 抑制剂 [抗 CRISPR (Acr) 蛋白] 的表达 当该平台通过 AAV 系统性递送至小鼠时，Acr-TS 被肝脏特异性 miRNA 靶向。 与组织特异性启动子不同，这种 Acr-TS 策略可以适应一种或多种。 只要肌肉特异性 (myo)-miRNA 能够抑制 Acr，DMD 就会影响多个肌肉组织。 在 Erik Sontheimer (CRISPR、Acr)、Eric Olson (DMD)、Wen Xu（体内 CRISPR 递送）、Phillip 的支持下 Zamore (miRNA)、Guangping Taka (AAV) 和zhiping Weng (生物信息学)，该提案旨在开发一种 治疗 DMD 的肌肉特异性编辑平台 myo-miRNA，miR-1，可以抑制肌肉细胞系中的 Acr。 为了实现肌肉特异性编辑，为了微调 DMD 肌肉组织中的编辑特异性，目标 1 将进行测试。 丰度和肌肉类型特异性不同的 myo-miR 抑制 Acr 和驱动肌肉特异性的能力 将提供支持最高肌肉特异性编辑的 myo-miR 构建体。 DMD 小鼠模型、体内肌肉功能以及肌营养不良蛋白外显子跳跃、肌营养不良蛋白蛋白、 目标2将测量肌肉组织和肝脏中的miRNA水平，测试额外Cas9的兼容性。 Acr-TS 系统中的直向同源物能够靶向更多序列，并开发单一 AAV 递送系统 为了提高安全性，已鉴定出抑制待测 Cas9 的 Acr 抑制 miR-1 的能力。 每个 Cas9 的 Acr 和驱动肌肉特异性编辑将在编码 Acr- 的单个载体中进行测试。 TS系统将被设计并封装到AAV中，肌肉特异性编辑将与双AAV进行比较 这项工作将开发一个灵活、安全、肌肉特异性的 CRISPR 平台，有潜力成为小鼠体内的系统。 用于任何肌肉组织组合来治疗 DMD 或其他遗传性肌肉疾病患者。