CRISPR/Cas9-based gene editing approaches for the treatment of USH2A-associated diseases

基于 CRISPR/Cas9 的基因编辑方法用于治疗 USH2A 相关疾病

基本信息

批准号：
10626831
负责人：
Qin Liu
金额：
$ 63.76万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10626831
关键词：
Abbreviations Affect Alternative Splicing Alternative Therapies Amino Acids Animals Antisense Oligonucleotides Bilateral Hearing Loss Biological Assay Biological Process Birth Blindness CRISPR/Cas technology Cell Line Cells Clinical Research Code Complement DNA Dependovirus Disease Europe Event Excision Exons Frameshift Mutation Gene Delivery Genes Genetic Genome Goals Human Individual Inherited Knockout Mice Mediating Medical Mendelian disorder Modality Mus Mutate Mutation Nonsense Mutation Open Reading Frames Organoids Orthologous Gene Pathogenicity Patients Persons Phase I/II Clinical Trial Phenotype Pilot Projects Proteins RNA Splicing Reading Frames Reporting Research Retina Retinitis Pigmentosa Safety Site Splice-Site Mutation System Technology Testing Therapeutic Transcript USH2A gene Usher Syndrome Usher Syndrome Type 2 Variant Viral Vision Work Zebrafish adeno-associated viral vector autosome base base editing base editor deaf emerging adult exon skipping exon skipping therapy gene augmentation therapy gene therapy genetic approach hearing impairment humanized mouse improved in vivo induced pluripotent stem cell inherited retinal degeneration innovative technologies intein interest mouse model mutant mutation correction novel therapeutics nuclease preclinical study prevent prevent hearing loss prime editing prime editor protein complex repaired success therapy development tool treatment strategy

项目摘要

Project Summary/Abstract Adeno-associated virus (AAV)-mediated gene augmentation is currently the most effective approach for treating recessive inherited retinal degenerations (IRDs). However, due to the limited capacity of AAV vectors, gene augmentation currently is not possible for one third of IRD patients due to mutations in genes that are too large for AAV, e.g., USH2A, which has a coding sequence of 15.6kb. Mutations in the USH2A gene are the leading cause of retinitis pigmentosa (RP) and Usher Syndrome Type II (USH2). These two diseases affect approximately 500,000 people worldwide. Both diseases are characterized by progressive vision loss beginning in early adulthood, with the addition of bilateral hearing loss from birth in USH2. Our long-term goal is to develop therapeutics that slow or prevent vision and hearing loss in USH2A patients. Over 1000 pathogenic and likely pathogenic variants have been identified in the USH2A gene. To date, studies have mainly focused on developing therapeutic strategies for the most prevalent mutation c.2299delG in exon 13 of the USH2A gene, which accounts for approximately one third of all USH2A cases in Europe and the U.S. c.2299delG disrupts the open reading frame of USH2A transcript. Multiple genetic approaches have been and are being tested to repair this genetic defect, including antisense oligonucleotide (ASO)-based exon skipping therapy or CRISPR/Cas9 gene editing. The exon skipping strategy have been extensively pursued in the past several years. Multiple lines of pre-clinical and clinical studies have demonstrated that excision of exon 13 from the USH2A gene can restore the disrupted open reading frame caused by c.2299delG mutation and lead to rescue of the retinal phenotypes in animals and improved vision in human. These findings strongly support the use of exon skipping as a treatment modality for some of the USH2A patients. The specific object of the proposed research is to investigate the potentials of innovative technologies, including exon skipping, base editing and prime editing, for the treatment of USH2A-related diseases. We plan to perform three proof-of- concept studies in three specific aims. In Aim 1, we will identify which other exons in the USH2A gene can serve as the next targets for exon skipping approach. To achieve this goal, we will generate mouse model that lacks an exon of interest and evaluate the ability of the abbreviated forms of the USH2A protein is sufficient to correct USH2A deficient phenotypes in mice. In Aim 2, we will explore the use of base editing to induce exon skipping in cells and human retinal explants, with the hope to circumvent the constraints of ASO, or Cas9 nuclease mediated exon skipping approaches. In Aim 3, we will investigate the feasibility of implementing prime editing to directly correct the mostly reported mutations in the USH2A gene, in particular the c.2299delG. If successful, these studies will open new therapeutic avenues for USH2A-associated disorders. It will also provide a template for similar efforts to develop therapies for other IRDs due to mutations in large genes.

项目概要/摘要腺相关病毒（AAV）介导的基因增强是目前最有效的方法治疗隐性遗传性视网膜变性（IRD）。然而，由于AAV载体的容量有限，由于基因突变太严重，目前三分之一的 IRD 患者无法进行基因增强治疗。 AAV 较大，例如 USH2A，其编码序列为 15.6kb。 USH2A 基因的突变是色素性视网膜炎 (RP) 和 II 型亚瑟综合症 (USH2) 的主要原因。这两种疾病影响全球约有 500,000 人。这两种疾病的特点是进行性视力丧失从成年早期开始，USH2 中还增加了出生后双侧听力损失。我们的长期目标旨在开发减缓或预防 USH2A 患者视力和听力损失的疗法。超过1000 USH2A 基因中已鉴定出致病性和可能致病性变异。迄今为止，研究已主要致力于针对 13 号外显子中最常见的突变 c.2299delG 制定治疗策略 USH2A 基因，约占欧洲和美国所有 USH2A 病例的三分之一 c.2299delG 破坏 USH2A 转录本的开放阅读框。多种遗传方法已经被正在测试修复这种遗传缺陷，包括基于反义寡核苷酸（ASO）的外显子跳跃疗法或 CRISPR/Cas9 基因编辑。外显子跳跃策略在过去被广泛采用几年。多项临床前和临床研究表明，外显子 13 的切除 USH2A基因可以恢复由c.2299delG突变引起的被破坏的开放阅读框并导致拯救动物的视网膜表型并改善人类的视力。这些发现有力地支持了使用外显子跳跃作为一些 USH2A 患者的治疗方式。其具体对象为拟议的研究旨在调查创新技术的潜力，包括外显子跳跃、碱基编辑和prime编辑，用于治疗USH2A相关疾病。我们计划进行三项证明- 三个具体目标的概念研究。在目标 1 中，我们将确定 USH2A 基因中的哪些其他外显子可以作为外显子跳跃方法的下一个目标。为了实现这一目标，我们将生成小鼠模型缺乏感兴趣的外显子并评估 USH2A 蛋白缩写形式的能力足以纠正小鼠中 USH2A 缺陷表型。在目标 2 中，我们将探索使用碱基编辑来诱导外显子跳过细胞和人类视网膜外植体，希望能够规避 ASO 或 Cas9 的限制核酸酶介导的外显子跳跃方法。在目标3中，我们将研究实施的可行性 Prime 编辑可直接纠正 USH2A 基因中最常见的突变，特别是 c.2299delG。如果成功，这些研究将为 USH2A 相关疾病开辟新的治疗途径。它还将为因大基因突变而开发其他 IRD 的类似努力提供了一个模板。