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

点击翻译按钮获取中文摘要

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.