evaluating the therapeutic potential of gene editing in a pig model with a dominant negative mutation in the GUCY2D gene

在SPP 2127的第一个资助期内，提供了种子资金，用于生成GUCY 2D中显性负突变的猪模型，导致一种形式的视锥-视杆变性（CORD 6）。我们利用该方法成功地在猪原代细胞中操纵了目的基因，并进行了初步的体细胞核移植实验。怀孕已经得到证实，这表明我们将在2021年底之前接收第一批创始动物。在这个项目中，我们将在这个模型中对表型进行初步表征，并开发一种基因编辑方法来治疗致病突变。表型分型将以多学科的方式进行，使用视网膜电图（ERG）、光学相干断层扫描（OCT）、视觉引导行为测试以及形态学和分子分析的组合。除了表型表征，我们的目标是定义治疗CORD 6的治疗窗口。为了治疗遗传病因，我们将遵循破坏性方法，即特异性破坏突变等位基因的开放阅读框架的基因编辑策略，以及重建方法，即修复突变并通过碱基编辑或引物编辑重建原始编码序列的基因编辑策略。此外，我们将研究将大型CRISPR/Cas组件包装到AAV载体中的机会，例如通过多个内含肽裂解系统，以定义一种有前途的治疗方法。在第三个工作包中，我们将通过单次注射AAV将最有前途的基因编辑策略应用于一组GUCY 2D猪，并通过行为测试和ERG和OCT检查的纵向监测来跟踪治疗效果6个月。实验结束后，将对眼球进行采样，并分析视网膜的治疗性基因编辑效果。我们在这个项目中的主要目标是研究基因编辑对显性阴性遗传性视网膜疾病的治疗潜力。此外，我们将了解锥杆变性的发病机制，并将建立一个繁殖群的模型，提供实验动物在我们自己的未来研究以及合作伙伴。

During the first funding period of SPP2127, a seed funding was provided to generate a pig model for a dominant negative mutation in the GUCY2D, causing a form of cone-rod degeneration (CORD6). We used the means to successfully manipulate the target gene in pig primary cells and to conduct initial somatic cell nuclear transfer experiments. Pregnancies have been confirmed, suggesting that we will receive first founder animals until end of 2021. Within this project we will perform an initial characterization of the phenotype in this model and develop a gene editing approach to treat the causative mutation. The phenotyping will be done in a multi-disciplinary way, using a combination of electroretinography (ERG), optical coherence tomography (OCT), visually guided behavior test as well as morphological and molecular analysis. In addition to phenotypical characterization, we aim at defining a therapeutic window for treating CORD6. For treating the genetic causative, we will follow disruptive approaches, i.e. gene editing strategies that specifically destroy the open reading frame of the mutated allels, as well as reconstituting approaches, i.e. gene editing strategies that repair the mutation and re-establish the original coding sequence by base editing or prime editing. In addition we will examine the opportunities to package the large CRISPR/Cas components into AAV-vectors, e.g. by multiple intein splitting systems, to define a promising therapeutic approach. In a third workpackage, we will apply the most promising gene editing strategy to a cohort of GUCY2D pigs by a single does injection of AAV and follow the effect of the treatment for 6 months by longitudinal monitoring with behavior tests and ERG and OCT examination. After terminating the experiments eye balls will be sampled and retina analysed for the efficacy of therapeutic gene editing. Our main goal in this project is to examine the therapeutic potential of gene editing for dominant negative inherited retinal diseases. In addition, we will get an idea of the pathogenesis of cone-rod degenerations and will establish a breeding herd for the model to provide experimental animals in our own future studies as well as for collaboration partners.