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

用于治疗斯塔加特病的治疗性基因组编辑 â 猪模型的生成和表型分析以及治疗方法的开发

• 批准号: 412915769
• 负责人: Professor Dr. Knut Stieger
• 金额: --
• 依托单位: DEP of Reproductive and Developmental Biology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/412915769?language=en
• 关键词: Therapeutic; genome; editing; treat; Stargardt

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.

由ABCA 4基因突变引起的Stargardt病是最常见的青少年黄斑病。该疾病最常在黄斑区域内表现为RPE和视锥光感受器变性，随后在后期阶段出现全身性视锥和视杆变性。ABCA 4蛋白通过感光盘膜运输类维生素A，从而使它们可用于进入视觉周期。蛋白质的缺失或功能缺失突变导致RPE内毒性类维生素A的积累，导致细胞死亡。由于缺乏合适的动物模型系统，使得科学家们无法收集更多关于早期视锥细胞感光疾病致病性的细节，也无法开发有效的治疗方法。使用高度特异性核酸内切酶（如CRISPR-Cas9系统）的治疗性基因组编辑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。理想地，通过使用包含野生型序列的模板DNA，可以在该修复过程中校正致病突变。不幸的是，很少有人知道的DNA修复活动在有丝分裂后和高度专业化的photoreceptors.The迷你猪代表了一个独特的，生物医学模型，由于其大小和可比的生理参数与男性，以及其视网膜形态与锥丰富的区域。为了研究ABCA 4相关的RPE和视锥光感受器疾病并开发治疗性基因组编辑作为治疗方法，我们的目标是产生含有人类病理性V1973 X无效突变的Stargardt病猪模型。为了实现这一目标，我们将加入跨学科团队。我们将首先在单细胞胚胎中通过CRISPR-Cas9介导的基因组编辑产生仔猪。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序列，类维生素A终产物定量）分析方法对其进行表型分析，并开始产生集落。我们将同时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。我们将在疾病模型中使用最佳组合之前，在野生型猪中研究AAV基因组编辑所需的所有组件的基因转移。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。我们的跨学科联合项目将在相关的大型动物模型中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。