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

Stargardt病是最常见的青少年黄斑病变，由ABCA4基因突变引起。该病最常表现在黄斑区，表现为视网膜色素上皮和视锥感光细胞变性，继而是晚期的全身性视锥和视杆细胞变性。ABCA4蛋白通过光感受器盘膜运输维甲酸，从而使它们可以进入视觉周期。蛋白质的缺失或功能突变的丧失会导致有毒的维甲酸在RPE内积聚，导致细胞死亡。由于缺乏合适的富含视锥细胞视网膜的动物模型系统，科学家们无法收集到更多关于早期视锥细胞光感受器疾病的致病机制的详细信息，也无法开发出有效的治疗方法。利用高度特异的内切酶进行基因组编辑，如CRISPR-Cas9系统，最近在基因治疗领域引起了极大的关注。它们在靶部位诱导双链断裂，随后由细胞自身的修复机制修复。理想情况下，通过使用包括野生型序列的模板DNA，致病突变可以在这个修复过程中得到纠正。不幸的是，人们对有丝分裂后和高度专业化的光感受器的DNA修复活动知之甚少。由于小型猪的大小和与男性相似的生理参数，以及它的视网膜形态和锥体丰富的区域，它代表了一种独特的生物医学模型。为了研究ABCA4相关的RPE和视锥感光细胞疾病，并开发治疗性基因组编辑作为治疗方法，我们的目标是建立一种含有人类病理性V1973X零突变的Stargardt病猪模型。为了实现这一目标，我们将联合起来组建一个跨学科的团队。我们将首先通过CRISPR-Cas9介导的单细胞胚胎基因组编辑来产生仔猪。一旦成功制作出创始动物，我们将通过体内(视网膜电描记术、光学相干断层扫描、行为研究)以及死后(视网膜形态、超形态、免疫组织化学、蛋白质组学、RNA序列、视黄醇最终产物定量)分析方法对它们进行表型鉴定，并开始产生菌落。我们将同时研究猪光感受器的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。在疾病模型中采用最佳成分之前，我们将研究AAV对野生型猪基因组编辑所需的所有成分的基因转移。来自该项目的数据将使我们能够在后续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和安全的治疗应用，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.