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.

项目摘要 Duchenne肌肉营养不良（DMD） - 致命的遗传性肌营养不良症 - 是由肌营养不良蛋白丧失引起的 维持肌肉完整性的蛋白质。皮质类固醇缓慢DMD进展，但会导致副作用。 解决DMD的根本原因可以改善患者健康，而无需皮质类固醇。许多DMD- 导致突变破坏肌营养不良蛋白mRNA阅读框，导致非功能蛋白。策略 跳过插图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 在肌营养不良蛋白mRNA中选择外显子，但需要终身输注，仅在一小组患者中起作用。使用 CRISPR编辑肌营养不良蛋白只需要一种治疗。 CRISPR介导的剪接站点的消融 外显子跳动可以增加DMD模型中的肌营养不良蛋白。但是，在意外组织中进行编辑是一个安全问题 CAS9疗法。 DMD的理想平台将限制编辑肌肉组织以最大程度地提高治疗益处。 实现组织特异性编辑的努力通常依赖于与组织的腺相关病毒（AAV）的递送 向性;但是，这很少是绝对的。最近，使用组织特异性miRNA来实现组织特异性编辑 通过miRNA靶位点（TS）在3'UTR中调节CAS9抑制剂[抗crispr（ACR）蛋白]的表达 ACR mRNA。当平台通过AAV系统地传递到小鼠时，由肝脏特异性miRNA靶向的ACR-TS 仅允许在肝脏中进行编辑。与组织特异性启动子不同，此ACR-TS策略可以适用于一个或 只要肌肉特异性（myo）-MIRNA可以抑制ACR，多种DMD中受影响的肌肉组织。 在Erik Sontheimer（CRISPR，ACR），Eric Olson（DMD）的支持下 Zamore（mirna），uganging gao（AAV）和Zhiping Weng（生物信息学），该提议旨在发展一个 治疗DMD的肌肉特异性编辑平台。 myo-miRNA miR-1可以在肌肉细胞系中抑制ACR 获得特定于肌肉的编辑。为了微调DMD肌肉时间的编辑特异性，AIM 1将测试 肌电肌的能力在丰度和肌肉类型的特异性方面有所不同，以压抑ACR并驱动肌肉特异性 在小鼠细胞系中进行编辑。将交付支持最高肌肉特异性编辑的Myo-Mir构造 到DMD小鼠模型，体内肌肉功能以及肌营养不良蛋白外显子跳动，肌营养不良蛋白， 将测量肌肉组织和肝脏中的miRNA水平。 AIM 2将测试其他CAS9的兼容性 ACR-TS系统中的直系同源物可以实现更多序列，并开发单个AAV输送系统 为了提高安全性。已经确定了抑制要测试的CAS9的ACR。 mir-1复制的能力 每个CAS9的ACR和驱动肌肉特异性编辑将在细胞中测试。编码ACR-的单个向量 TS系统将被设计并包装到AAV中，并将特定于肌肉的编辑与双AAV进行比较 小鼠的系统。这项工作将开发一个灵活，安全，特定于肌肉的CRISPR平台，并有可能成为 用于治疗DMD或其他遗传肌肉疾病患者的任何组合。