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

在SPP2127的第一个资助期，提供了一笔种子资金，用于生成GUCY2D显性负突变的猪模型，该突变导致锥杆变性（CORD6）。我们利用该方法成功地在猪原代细胞中操纵靶基因，并进行了初始体细胞核移植实验。怀孕已经确认，这意味着我们将在2021年底之前收到第一批创始动物。在这个项目中，我们将对该模型中的表型进行初步表征，并开发一种基因编辑方法来治疗致病突变。表型分析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相结合。除了表型表征外，我们的目标是确定治疗CORD6的治疗窗口。对于治疗遗传致病，我们将采用破坏性方法，即专门破坏突变等位基因开放阅读框的基因编辑策略，以及重构方法，即通过碱基编辑或引体编辑修复突变并重新建立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，例如通过多个内部分裂系统，以确定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将最有希望的基因编辑策略应用于一组GUCY2D猪，并通过行为测试、ERG和OCT检查进行纵向监测，跟踪治疗效果6个月。实验结束后，将对眼球进行采样，并对视网膜进行分析，以确定治疗性基因编辑的有效性。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，并为该模型建立一个繁殖群，为我们自己未来的研究提供实验动物，也为合作伙伴提供实验动物。

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.