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

项目摘要 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的缺失引起的， 一种维持肌肉完整性的蛋白质皮质类固醇减缓DMD进展但引起副作用。 解决DMD的根本原因可能会改善患者的健康，而不需要皮质类固醇。很多DMD- 导致突变破坏肌营养不良蛋白mRNA的阅读框架，产生无功能蛋白。战略 跳过框外外显子以恢复阅读框并产生半功能性蛋白质， 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，但需要终身输注，并且仅在一小群患者中起作用。使用 CRISPR编辑肌营养不良蛋白只需要一次治疗。CRISPR介导的剪接位点消融导致 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的理想平台将限制编辑肌肉组织，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）与组织一起递送。 但它很少是绝对的。组织特异性编辑最近使用组织特异性miRNA实现， 通过miRNA靶位点（TS）调节Cas9抑制剂[抗CRISPR（Acr）蛋白]的表达， Acr mRNA。当该平台通过AAV系统地递送给小鼠时，肝脏特异性miRNA靶向的Acr-TS 仅允许在肝脏中编辑。与组织特异性启动子不同，这种Acr-TS策略可以适用于一个或多个启动子。 DMD中受影响的多个肌肉组织，只要肌肉特异性（myo）-miRNA可以抑制Acr。 在Erik Sontheimer（CRISPR，Acr），Eric Olson（DMD），Wen Xue（体内CRISPR递送），菲利普（Phillip） Zamore（miRNA），Guangping Gao（AAV）和Zhiping Weng（生物信息学），该提案旨在开发一种新的方法， 肌肉特异性编辑平台来治疗DMD。myo-miRNA，miR-1，可以抑制肌肉细胞系中的Acr 以实现肌肉特定编辑。为了微调DMD肌肉组织中编辑的特异性，Aim 1将测试 在丰度和肌肉类型特异性方面变化的myo-miR抑制Acr和驱动肌肉特异性 在小鼠细胞系中进行编辑。支持最高肌肉特异性编辑的myo-miR构建体将被递送 DMD小鼠模型，以及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， 并测量肌肉组织和肝脏中的miRNA水平。目标2将测试额外的Cas9的兼容性 Acr-TS系统中的直系同源物，以能够靶向更多序列，并开发单一AAV递送系统 以提高安全性。已经鉴定了抑制待测试的Cas9的Acr。miR-1抑制 将在细胞中测试每个Cas9的这种Acr和驱动肌肉特异性编辑。编码Acr的单个载体- TS系统将被设计并包装到AV中，肌肉特定编辑将与双AV进行比较 系统在小鼠。这项工作将开发一种灵活，安全，肌肉特异性的CRISPR平台，有可能成为 用于肌肉组织的任何组合，以治疗DMD或其他遗传性肌肉疾病患者。

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.