Evaluation of novel gene therapy approaches for USH2A

Usher综合征（USH）的特征是由视网膜色素变性和先天性或进行性耳聋引起的视网膜缺陷，偶尔伴有前庭功能障碍。USH 2A基因突变是USH综合征最常见的原因，但目前还没有治疗方法。由于USH 2A基因的大小，使用金标准腺相关病毒（AAV）载体的经典基因补充疗法需要至少四个载体（四重AAV载体策略），并且尚不清楚这种方法原则上是否有效。在本项目中，为了建立USH 2A的适当治疗策略，我们将采用三种方法：i）使用引物和碱基编辑器的CRISPR/Cas介导的基因组编辑，以校正USH 2A中三种最常见的突变（c.2299delG和c.2276G>T）ii）使用mRNA反式剪接校正突变USH 2A转录物独立的四重AAV载体策略，使用在我们的初步工作中开发的方法在转录物水平上重建分裂的基因片段。所有方法将首先在适合各自治疗策略的不同人类细胞系中进行测试和优化。体外建立的程序随后将在人视网膜类器官和（如果可用）来自人供体视网膜的外植体中进一步验证。另外，将在转录物和蛋白质水平上研究USH 2A在使用突变非依赖性四重AAV载体策略注射的野生型小鼠和猪的视网膜中的表达和定位。此外，我们还将使用ush 2a斑马鱼模型和人源化c.2299delG小鼠模型研究四重AAV载体策略的结构和功能效应。最后，这种小鼠模型也将允许我们分析体内主要编辑策略的治疗成功。如果成功，这些实验将为未来使用创新技术的USH 2A临床试验铺平道路，这将使全球许多患者受益。

Usher syndrome (USH) is characterized by retinal defects due to retinitis pigmentosa and congenital or progressive deafness with occasional vestibular dysfunction. Mutations in the USH2A gene are by far the most common cause of USH syndrome, but there is currently no therapy. Because of the size of the USH2A gene, classical gene-supplementation therapies using the gold standard adeno-associated viral (AAV) vectors require at least four vectors (quadruple AAV vector strategy), and it is not clear whether this approach works in principle. In this project, to establish appropriate therapeutic strategies for USH2A, we will pursue three approaches: i) CRISPR/Cas-mediated genome editing using prime and base editors to correct three most common mutations in USH2A (c.2299delG and c.2276G>T) ii) Correction of mutant USH2A transcripts using mRNA trans-splicing iii) A mutation-independent quadruple AAV vector strategy using a method developed in our preliminary work to reconstitute split gene fragments at the transcript level. All approaches will first be tested and optimized in different human cell lines suitable for the respective therapeutic strategies. The procedures established in vitro will subsequently be further validated in human retinal organoids and (if available) in explants from human donor retinas. The expression and localization of USH2A will additionally be investigated in the retina of wild-type mice and pigs injected using the mutation-independent quadruple AAV vector strategy at the transcript and protein levels. In addition, we will also investigate the structural and functional effects of the quadruple AAV vector strategy using the ush2a zebrafish model and a humanized c.2299delG mouse model. Finally, this mouse model will also allow us to analyze the therapy success of the prime editing strategy in vivo. If successful, these experiments will pave the way for future USH2A clinical trials using innovative technologies that would benefit many patients worldwide.