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

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AV来说很大，例如，USH2A基因全长15.6kb。USH2A基因的突变是 视网膜色素变性（RP）和Usher综合征II型（USH2）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，在USH2中增加了出生时的双侧听力损失。我们的长期目标 是开发减缓或预防USH2A患者视力和听力损失的治疗方法。超过1000 在USH2A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 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（ASO）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， USH2A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USH2A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USH2A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USH2A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靶点。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USH2A蛋白的缩写形式的能力足以 在小鼠中纠正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 在细胞和人类视网膜外植体中跳跃，希望绕过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 引物编辑以直接校正USH2A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USH2A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。

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.