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

项目摘要/摘要 常染色体显性遗传性面肩臂肌营养不良症(FSHD)是最常见的肌肉营养不良症之一 营养不良，每7,500人中就有1人受到影响，每20,000人中就有1人受到影响。FSHD被正式归类为主要形式的 肌肉营养不良是在1954年，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的发病机制与DUX4基因的异常表达有关， 它编码一种肌毒素转录因子。DUX4的出现是FSHD背后的主要侮辱 代表了该领域的势头转变，因为它为模型开发和治疗提供了重要的目标 设计。事实上，由于FSHD目前无法治疗，开发有效的FSHD疗法是 菲尔德。我们假设FSHD治疗的中心应该是抑制骨骼中有毒的DUX4表达 肌肉。这项建议的目标是开发有效的前瞻性FSHD疗法，旨在减少或 消除有毒的DUX4表达。为此，我们将使用新的尖端RNA静默和RNA编辑 在过去2-3年内才出现技术的方法。这些方法只能切割RNA和 而不是DNA，因此不会对宿主细胞造成永久性基因组修改的风险。因为这些策略 它们只是新出现的，从未在动物模型中使用基因治疗方法在体内进行过测试 疾病的威胁。我们将在这里这样做，因此我们的建议将为体内疗效的使用提供第一个原则证明 CRISPR-Cas13和RNA编辑技术。具体地说，在目标1中，我们将开发一个新的CRISPR/Cas13 基因治疗的方法是在DUX4 mRNA可以被制成蛋白质之前将其裂解。在目标2中，我们将开发RNA 编辑策略，旨在改变DUX4成绩单，使其无法产生有毒的完整- DUX4蛋白的长度形式。最后，由于这些战略是新的，而且大量的非目标数据目前 缺乏，在目标3，我们将评估RNA切割和编辑方法在人类FSHD和 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，支持 翻译最终可用于翻译的基于AAV的RNA靶向治疗FSHD的新疗法 朝着我们的临床应用目标迈进。这些数据还可能支持该技术在其他领域的更广泛使用 遗传性疾病。

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.