Evaluation of novel gene therapy approaches for USH2A

Usher综合征（USH）的特点是视网膜缺损，由于视网膜色素变性和先天性或进行性耳聋，偶尔前庭功能障碍。到目前为止，USH2A基因突变是USH综合征最常见的原因，但目前尚无治疗方法。由于USH2A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（四倍AAV载体策略），并且尚不清楚该方法是否原则上有效。在本项目中，为了建立合适的USH2A治疗策略，我们将采用三种方法：i) CRISPR/ cas介导的基因组编辑，使用引物和碱基编辑器纠正USH2A中最常见的三种突变（c.2299delG和c.2276G>T） ii)使用mRNA反式剪接纠正突变的USH2A转录本iii)使用我们初步工作中开发的方法，采用突变无关的四倍AAV载体策略，在转录水平上重建分裂的基因片段。所有的方法都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。在体外建立的程序随后将在人类视网膜类器官和（如果有的话）人类供体视网膜的外植体中进一步验证。此外，我们还将在使用不依赖突变的四倍AAV载体策略注射的野生型小鼠和猪的视网膜中研究USH2A在转录物和蛋白质水平上的表达和定位。此外，我们还将利用ush2a斑马鱼模型和人源化c.2299delG小鼠模型来研究四联AAV载体策略的结构和功能效应。最后，这个小鼠模型也将允许我们分析启动编辑策略在体内的治疗成功。如果成功，这些实验将为未来使用创新技术的USH2A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.