CRISPR-Cas13 gene therapy and RNA editing for Facioscapulohumeral muscular dystrophy (FSHD)

项目概要/摘要 常染色体显性面肩肱型肌营养不良症（FSHD）是最常见的肌营养不良症之一， 营养不良，影响1/7，500至1/20，000的个体。FSHD被正式归类为一种主要形式， 1954年，他患上了肌肉萎缩症，但导致这种疾病的致病事件直到最近才开始出现 聚焦如今，人们认识到FSHD的发病机制涉及DUX 4基因的异常表达， 其编码肌毒性转录因子。DUX 4作为FSHD基础的主要损伤的出现 代表了该领域的动力转变，因为它为模型开发和治疗提供了重要目标 设计事实上，由于FSHD目前无法治疗，因此开发有效的FSHD疗法是治疗FSHD的关键需求。 领域我们假设FSHD治疗应该集中在抑制骨骼肌中毒性DUX 4的表达， 肌肉.该提案的目的是开发有效的前瞻性FSHD疗法，旨在减少或 消除毒性DUX 4表达。为此，我们将使用新的尖端RNA沉默和RNA编辑技术， 这些方法的技术仅在过去2-3年内出现。这些方法仅切割RNA， 而不是DNA，因此不会对宿主细胞造成永久性基因组修饰的风险。因为这些策略 只是新出现的，它们从未在动物模型中使用基因治疗方法进行过体内测试 疾病。我们将在这里这样做，因此，我们的建议将提供第一个证明的原则，在体内的功效使用 CRISPR-Cas 13和RNA编辑技术。具体来说，在目标1中，我们将开发一种新的CRISPR/Cas 13 基因治疗方法切割DUX 4 mRNA，然后再将其制成蛋白质。在目标2中，我们将开发RNA 编辑策略，旨在改变DUX 4转录本，使其无法产生有毒的全， DUX 4蛋白的长度形式。最后，由于这些策略是新的，并且目前存在大量脱靶数据， 在目标3中，我们将评估RNA切割和编辑方法在人FSHD中的精确性， 控制肌肉细胞。在完成这些目标后，我们预计将产生临床前数据， 用于FSHD的新的基于AAV的RNA靶向疗法的翻译， 朝着我们临床应用的目标前进。这些数据也可能支持这项技术在其他领域的更广泛应用。 遗传疾病

Project Summary/Abstract Autosomal dominant Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent muscular dystrophies, affecting 1 in 7,500 to 1 in 20,000 individuals. FSHD was formally classified as a major form of muscular dystrophy in 1954, but the pathogenic events leading to the disease have only recently started coming into focus. Today, it is now recognized that FSHD pathogenesis involves aberrant expression of the DUX4 gene, which encodes a myotoxic transcription factor. The emergence of DUX4 as a primary insult underlying FSHD represented a momentum shift in the field as it provided an important target for model development and therapy design. Indeed, as FSHD is currently untreatable, developing effective FSHD therapies is a critical need in the field. We hypothesized that an FSHD treatment should center on inhibiting toxic DUX4 expression in skeletal muscles. The objective of this proposal is to develop effective prospective FSHD therapies aimed at reducing or eliminating toxic DUX4 expression. To do this, we will use new cutting edge RNA silencing and RNA editing approaches for which technology emerged only within the last 2-3 years. These approaches only cut RNA and not DNA, and therefore pose no risk of permanent genome modification in host cells. Because these strategies are only newly emerged, they have never been tested in vivo using gene therapy approaches in an animal model of disease. We will do that here, and thus our proposal will provide first proof-of-principle for in vivo efficacy use of CRISPR-Cas13 and RNA editing technology. Specifically, in Aim 1 we will develop a new CRISPR/Cas13 gene therapy approach to cleave DUX4 mRNA before it can be made into protein. In Aim 2, we will develop RNA editing strategies that are designed to change the DUX4 transcript and render it incapable of producing toxic full- length forms of DUX4 protein. Finally, since these strategies are new and abundant off-target data are currently lacking, in Aim 3, we will assess the precision of RNA cleavage and editing approaches in human FSHD and control muscle cells. Upon completion of these Aims, we expect to produce pre-clinical data supporting the translation of new AAV-based RNA-targeted therapies for FSHD that can be ultimately used for translation toward our goal of clinical application. These data may also support the broader use of this technology for other genetic disorders.