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

• 批准号: 10446571
• 负责人: Qin Liu
• 金额: $ 63.51万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 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/10446571
• 关键词: 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; GTP-Binding Protein alpha Subunits, Gs; Gene Delivery; Genes; Genetic; Genome; Goals; Human; Inherited; Knockout Mice; Lead; 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; 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

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.

项目摘要/摘要 腺相关病毒（AAV）介导的基因增强目前是目前最有效的方法 治疗隐性遗传性视网膜变性（IRD）。但是，由于AAV向量的能力有限， 由于基因的突变也是如此 AAV的大型，例如USH2A，其编码顺序为15.6kb。 USH2A基因中的突变是 视网膜炎色素炎（RP）和USHER综合征II型（USH2）的主要原因。这两种疾病影响 全球约有50万人。两种疾病的特征都是渐进视力丧失 从成年初开始，随着USH2出生时增加双侧听力损失。我们的长期目标 是为了开发缓慢或防止USH2A患者视力和听力损失的治疗剂。超过1000 在USH2A基因中已经鉴定出致病性和可能的​​致病变异。迄今为止，研究有 主要专注于为最普遍的突变开发治疗策略C.2299delg在外显子13中 USH2A基因约占欧洲和美国所有USH2A病例的三分之一 c.2299delg破坏了USH2A成绩单的开放阅读框架。多种遗传学方法已经过去了 正在测试以修复这种遗传缺陷，包括反义寡核苷酸（ASO）跳过的外显子跳过 治疗或CRISPR/CAS9基因编辑。过去的外显子跳过策略已被广泛采用 几年。临床前和临床研究的多种线表明，从外显子13切除 USH2A基因可以恢复由C.2299delg突变引起的破坏的开放阅读框架，并导致 营救动物中的视网膜表型，并改善人类的视力。这些发现强烈支持 使用外显子跳过作为某些USH2A患者的治疗方式。特定对象 拟议的研究是研究创新技术的潜力，包括外显子跳过，基础 编辑和主要编辑，用于治疗USH2A相关疾病。我们计划执行三个证明 概念研究以三个具体目标。在AIM 1中，我们将确定USH2A基因中哪些其他外显子可以 作为外显子跳过方法的下一个目标。为了实现这一目标，我们将生成鼠标模型 缺乏感兴趣的外显子并评估USH2A蛋白缩写形式的能力足以使 纠正小鼠中USH2A缺乏表型。在AIM 2中，我们将探索使用基础编辑来诱导外显子的使用 跳过细胞和人类视网膜外植体，希望避免ASO的约束或Cas9的约束 核酸酶介导的外显子跳过方法。在AIM 3中，我们将调查实施的可行性 素数编辑以直接纠正USH2A基因中的大多数突变，尤其是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。它也会 为由于大基因突变而为其他IRD的疗法提供类似努力的模板。