Therapeutic genome editing to treat Stargardt disease – Generation and phenotyping of a porcine model and development of a treatment approach

由ABCA4基因突变引起的Stargardt病是最常见的青少年黄斑病。该病最常在黄斑区域表现为RPE和锥状光感受器变性，晚期继发广泛性锥状和杆状变性。ABCA4蛋白通过光感受器圆盘膜运输类维甲酸，从而使它们能够进入视觉循环。蛋白质缺失或功能突变丧失会导致毒性类维生素a在RPE内积聚，导致细胞死亡。缺乏具有丰富视锥细胞视网膜的适当动物模型系统，阻碍了科学家收集更多关于早期视锥细胞光感受器疾病致病性的细节，并开发有效的治疗方法。利用高度特异性内切酶的治疗性基因组编辑，如CRISPR-Cas9系统，最近在基因治疗领域引起了广泛关注。它们在靶位点诱导双链断裂，随后由细胞自身的修复机制修复。理想情况下，通过使用包含野生型序列的模板DNA，可以在此修复过程中纠正致病突变。不幸的是，人们对有丝分裂后和高度特化的光感受器的DNA修复活性知之甚少。这只迷你猪代表了一种独特的生物医学模型，这要归功于它的大小和与人类相似的生理参数，以及它的视网膜形态与锥体丰富的区域。为了研究ABCA4相关的RPE和锥体光感受器疾病，并开发治疗性基因组编辑作为治疗方法，我们的目标是生成含有人类病理性V1973X零突变的Stargardt病猪模型。为了实现这一目标，我们将在一个跨学科的团队中共同努力。我们将首先通过CRISPR-Cas9介导的基因组编辑在一个细胞胚胎中产生仔猪。在成功生产创始动物后，我们将通过体内（视网膜电图，光学相干断层扫描，行为研究）以及死后（视网膜形态学，超形态学，免疫组织化学，蛋白质组学，RNA测序，类视黄醇终产物定量）分析方法对它们进行表型分析，并将开始产生一个菌落。我们将平行研究猪光感受器中的DNA修复活性，并通过适当干扰DNA修复蛋白来优化基因组编辑活性。我们将研究AAV在野生型猪中进行基因组编辑所需的所有成分的基因转移，然后在疾病模型中使用最佳成分。这个项目的数据将使我们在后续的研究中继续优化治疗方法，以及研究其他治疗方法，如细胞移植。我们的联合跨学科项目将在相关的大型动物模型上开发一种高效、安全的治疗应用，为未来的临床应用铺平道路。

Stargardt disease, caused by mutations in the ABCA4 gene, is the most common juvenile maculopathy. The disease manifests most often within the macular area with RPE and cone photoreceptor degeneration, succeeded by generalized cone and rod degeneration at later stages. The ABCA4 protein transports retinoids through the photoreceptor disc membrane, thus making them available for entering the visual cycle. Absence of the protein or loss of function mutations cause accumulation of toxic retinoids within the RPE, leading to cell death. Absence of appropriate animal model systems with cone enriched retinae has prevented scientists from gathering more details about the pathogenicity for early cone photoreceptor disease and to develop effective treatment approaches.Therapeutic genome editing employing highly specific endonucleases, such as the CRISPR-Cas9 system, has recently gathered much attention in the gene therapy field. They induce a double strand break at the target site, which is subsequently repaired by the cells own repair mechanisms. Ideally, through the use of a template DNA comprising the wild type sequence, a disease-causing mutation can be corrected during this repair process. Unfortunately, little is known about DNA repair activity in post-mitotic and highly specialized photoreceptors.The minipig represents a unique, biomedical model thanks to its size and comparable physiological parameters with men, as well as its retinal morphology with a cone enriched region. To study ABCA4 associated RPE and cone photoreceptor disease and to develop therapeutic genome editing as treatment approach, we aim at generating a porcine model of Stargardt disease containing the human pathologic V1973X null mutation. To achieve this goal, we will join forces in an interdisciplinary team. We will first generate piglets by CRISPR-Cas9 mediated genome editing in one cell embryos. Upon successful production of founder animals, we will phenotype them by in vivo (electroretinography, optical coherence tomography, behavioral studies) as well as post mortem (retinal morphology, ultra-morphology, immunohistochemistry, proteomics, RNA seq, retinoid end product quantification) analysis methods, and will start generating a colony. We will in parallel study DNA repair activity in porcine photoreceptors and optimize genome editing activity by appropriate interference with DNA repair proteins. We will study gene transfer of all components necessary for genome editing by AAV in wild type pigs prior to employing the optimal composition in the disease model.Data from this project will enable us to continue optimizing the treatment approach in subsequent studies, as well as studying other treatment approaches such as cell transplantation. Our joint interdisciplinary project will develop an efficient and safe therapeutic application in a relevant large animal model that will pave the way towards clinical application in the future.