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

项目总结 Duchenne肌营养不良症(DMD)是一种致命的遗传性肌营养不良症，由肌营养不良蛋白的丢失引起， 一种维持肌肉完整性的蛋白质。皮质类固醇延缓了DMD的进展，但会产生副作用。 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。许多DMD- 引起突变扰乱了dystrophin mRNA的阅读框架，导致非功能蛋白的产生。策略 跳过框外外显子，恢复阅读框，产生半功能蛋白进行改良 肌肉功能可纠正%的DMD突变。FDA批准的反义寡核苷酸药物可以跳过 选择dystrophin mRNA中的外显子，但需要终生输注，并且只在一小部分患者中起作用。vbl.使用 CRISPR编辑dystrophin只需要一次治疗。CRISPR介导的剪接位点消融引起 在DMD模型中，外显子跳跃可以增加Dstrophin的表达。然而，在非预期的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益。 实现组织特异性编辑的努力通常有赖于腺相关病毒(AAV)对组织的传递 取向；然而，它很少是绝对的。最近使用组织特异性miRNAs实现了组织特异性编辑 通过miRNA靶点(TS)调控CAS9抑制剂[抗CRISPR(ACR)蛋白]在3‘非编码区的表达 ACR基因的表达。当该平台通过AAV系统地传递给小鼠时，ACR-TS被肝脏特异性miRNA靶向 仅允许在肝脏中进行编辑。与组织特异性启动子不同，这种ACR-TS策略可以适用于一种或多种 在DMD中，只要肌肉特异性(Myo)-miRNA可以抑制ACR，就会影响多个肌肉组织。 在埃里克·桑特海默(CRISPR，ACR)、埃里克·奥尔森(DMD)、文雪(体内CRISPR交付)、菲利普的支持下 扎莫尔(MiRNA)、高广平(AAV)和翁志平(生物信息学)，这项提议寻求开发一种 专门用于治疗DMD的肌肉编辑平台。Myo-miRNA，miR-1，可以抑制肌肉细胞系中的ACR 以实现特定肌肉的编辑。为了微调肌肉组织中编辑DMD的特异性，Aim 1将测试 不同丰度和肌肉类型特异性的myo-miRs抑制ACR和驱动肌肉特异性的能力 在小鼠细胞系中编辑。将提供支持最高肌肉特定编辑的myo-miR结构 到DMD小鼠模型，体内肌肉功能以及dystrophin外显子跳跃，dystrophin蛋白， 并将测量肌肉组织和肝脏中的miRNA水平。AIM 2将测试额外的Cas9的兼容性 ACR-TS系统中的同源基因，以实现更多序列的靶向，并开发单一的AAV递送系统 以提高安全性。已经确定了一种抑制Cas9被测试的ACR。MiR-1的抑制能力 由每个Cas9进行的ACR和驱动肌肉特定编辑将在细胞中进行测试。编码ACR的单个载体- TS系统将被设计并打包到AAV中，肌肉特定编辑将与双重AAV进行比较 系统在小鼠体内。这项工作将开发一个灵活、安全、针对肌肉的CRISPR平台，有可能成为 用于肌肉组织的任何组合，用于治疗DMD患者或其他遗传性肌肉疾病。

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.