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

面肩肱型肌营养不良症 (FSHD) 的 CRISPR-Cas13 基因治疗和 RNA 编辑

基本信息

批准号：
10469571
负责人：
Scott Q Harper
金额：
$ 60.79万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-15 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10469571
关键词：
4q35 Adenosine Affect Animal Disease Models Arginine CRISPR/Cas technology Cell Line Cells Chimeric Proteins Chromosomes Chronic Clinical Data Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Cytidine DNA Data Deamination Disease Effectiveness Engineering Enzymes Event Facioscapulohumeral Muscular Dystrophy Gene Expression Genes Genetic Diseases Genetic Transcription Genome Glutamine Goals Guanosine Human In Vitro Individual Initiator Codon Inosine Lead Length Location Mammals Mediating Messenger RNA Methods Mitotic Modeling Modification Molecular Mus Muscle Muscle Cells Muscular Dystrophies Mutate Myopathy Neuromuscular Diseases Other Genetics Pathogenesis Pathogenicity Persons Phenotype Proteins Publishing RNA RNA Editing RNA Interference RNA-targeting therapy Repression Ribosomes Risk Safety Side Skeletal Muscle Specificity System Technology Terminator Codon Testing Therapeutic Transcript Translations Uridine Work adeno-associated viral vector adenosine deaminase base base editing clinical application design effective therapy gene therapy genome editing improved in vivo in vivo evaluation model development muscular dystrophy mouse model novel strategies pre-clinical prospective recruit side effect targeted treatment therapeutic genome editing therapy design transcription factor transcriptome sequencing

项目摘要

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.

项目摘要/摘要常染色体显性骨膜瘤肌肉营养不良（FSHD）是最普遍的肌肉营养不良，影响20,000人中的7,500至1个。 FSHD被正式归类为主要形式 1954年的肌肉营养不良，但是导致该疾病的致病事件直到最近才开始集中精力。如今，现在已经认识到FSHD发病机理涉及Dux4基因的异常表达，它编码肌毒性转录因子。 DUX4作为主要侮辱的FSHD的出现代表该领域的动量转变，因为它为模型开发和治疗提供了重要目标设计。确实，由于FSHD目前不可处理，因此开发有效的FSHD疗法是至关重要的场地。我们假设FSHD治疗应集中于抑制骨骼中有毒Dux4的表达肌肉。该提案的目的是开发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沉默和RNA编辑技术仅在过去2 - 3年内出现的技术。这些方法只切断RNA，不是DNA，因此在宿主细胞中没有永久性基因组修饰的风险。因为这些策略只有新出现，它们从未在动物模型中使用基因疗法在体内进行测试疾病。我们将在这里这样做，因此我们的建议将为体内功效提供第一个原则证明 CRISPR-CAS13和RNA编辑技术。具体来说，在AIM 1中，我们将开发新的CRISPR/CAS13 在将其裂解DUX4 mRNA之前，可以将其制成蛋白质。在AIM 2中，我们将开发RNA 编辑策略旨在更改DUX4转录本并使其无法产生有毒的全部功能 DUX4蛋白的长度形式。最后，由于这些策略是当前的新且丰富的脱靶数据在AIM 3中，我们将评估人类FSHD和控制肌肉细胞。完成这些目标后，我们希望产生支持该目标的临床前数据最终可用于翻译的FSHD的新的基于AAV的RNA靶向疗法的翻译实现我们的临床应用目标。这些数据也可能支持该技术更广泛地用于其他遗传疾病。