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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.

项目总结/摘要腺相关病毒（AAV）介导的基因扩增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载体的能力有限，目前，三分之一的IRD患者不可能进行基因扩增，因为基因突变太对于AAV来说，例如，USH 2A基因全长15.6kb。USH 2A基因的突变是视网膜色素变性（RP）和Usher综合征II型（USH 2）的主要原因。这两种疾病影响全球约50万人。这两种疾病的特点是进行性视力下降从成年早期开始，加上USH 2出生时双侧听力损失。我们的长期目标是开发减缓或预防USH 2A患者视力和听力损失的治疗方法。超过1000 在USH 2A基因中已经鉴定出致病性和可能致病的变体。迄今为止，研究表明，主要集中在开发治疗策略的最流行的突变c.2299delG外显子13的 USH 2A基因，约占欧洲和美国所有USH 2A病例的三分之一。 c.2299delG破坏了USH 2A转录本的开放阅读框架。多种遗传学方法已经被采用，正在测试修复这种遗传缺陷，包括基于反义寡核苷酸（阿索）的外显子跳跃 CRISPR/Cas9基因编辑。外显子跳跃策略在过去已经被广泛地采用几年多线临床前和临床研究表明，切除外显子13， USH 2A基因可以恢复由c.2299delG突变引起的被破坏的开放阅读框架，并导致拯救动物的视网膜表型和改善人类的视力。这些发现有力地支持了使用外显子跳跃作为一些USH 2A患者的治疗方式。的具体对象拟议的研究是调查创新技术的潜力，包括外显子跳读，碱基突变，编辑和主要编辑，用于治疗USH 2A相关疾病。我们计划进行三次验证- 三个具体目标的概念研究。在目标1中，我们将确定USH 2A基因中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，并且评估USH 2A蛋白的缩写形式的能力足以在小鼠中纠正USH 2A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在细胞和人类视网膜外植体中跳跃，希望绕过阿索或Cas9的限制核酸酶介导的外显子跳跃方法。在目标3中，我们将研究实施引物编辑以直接校正USH 2A基因中报道最多的突变，特别是c.2299delG。如果成功，这些研究将为USH 2A相关疾病开辟新的治疗途径。它还将为类似的努力提供了一个模板，以开发针对其他IRD的治疗方法，这些IRD是由于大基因突变引起的。