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

项目摘要/摘要 腺相关病毒(AAV)介导的基因扩增是目前最有效的治疗方法 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， 目前三分之一的IRD患者不可能进行基因增强，因为基因突变太过 对于AAV来说很大，例如USH2a，它具有15.6kb的编码序列。USH2A基因的突变是 导致视网膜色素变性(RP)和Usher综合征II型(USH2)的主要原因。这两种疾病会影响 全球约有500,000人。这两种疾病的特征都是进行性视力丧失。 从成年早期开始，伴随着USH2出生时的双侧听力损失。我们的长期目标 是开发延缓或预防USH2A患者视力和听力损失的疗法。超过1000 已在USH2A基因中鉴定出致病和可能致病的变异。到目前为止，研究已经 主要致力于开发最常见的突变c.2299delG的治疗策略 USH2A基因，约占欧洲和美国USH2A病例的三分之一。 C.2299delG打乱了USH2A转录本的开放阅读框架。已经有了多种遗传方法 正在测试以修复这种遗传缺陷，包括基于反义寡核苷酸(ASO)的外显子跳过 治疗或CRISPR/Cas9基因编辑。外显子跳过策略在过去得到了广泛的研究 好几年了。多项临床前和临床研究表明，外显子13的切除 USH2A基因可修复由c.2299delG突变引起的开放阅读框中断，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 部分USH2A患者使用外显子跳跃作为一种治疗方式。对象的特定对象 拟议的研究是为了调查创新技术的潜力，包括外显子跳过、碱基 编辑和原文编辑，用于治疗USH2A相关疾病。我们计划执行三项证明- 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 作为外显子跳过方法的下一个靶点。为了实现这一目标，我们将生成 缺乏感兴趣的外显子，并评估USH2A蛋白的缩写形式足以 纠正小鼠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 启动编辑以直接纠正大多数已报道的USH2A基因突变，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 为开发其他因大基因突变而导致的IRD的治疗方法提供了一个模板。

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.