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.

项目总结