Development of Swine Reporter Models for Testing Somatic Cell Genome Editing Tools

项目摘要 精确的基因失活、基因添加和患者细胞中的基因修复都是新范例的一部分 基因治疗然而，这些基因编辑疗法的全部潜力只有在 可以直接治疗患者体内的每个组织。这一目标需要新的基因编辑 其中摄取动力学、渗透和分布良好的递送媒介物和给药途径 确立了习实现这一目标的第一步是开发模拟大小，生理学， 人类患者的衰老。猪模型符合这些标准，可以在子宫内，儿科和成人进行基因编辑 治疗因此，遗传学家一直在使用基因编辑来创建一套猪模型， 在过去的6年里，人类疾病等位基因的研究，以加强临床前的研究和开发。虽然这些 模型在有效性研究中有价值，这些动物的结果并不广泛适用于优化 体内编辑。为了解决这一限制，我们建议开发一套猪模型和载体， 报告基因编辑结果与体内（整个动物）和单细胞读数的组合。我们 模块化设计和生产流水线将使大多数人类疾病等位基因的快速和可靠的建模成为可能 在一个单一的报告系统。 拟议的模型将报告两种类型的基因编辑工具的结果：1）那些切割或切口 刺激DNA修复，即。CRISPR/Cas9、TALEN、ZFN，以及2）碱基编辑器， DNA碱基与靶位点的另一个碱基连接，而不产生双链断裂。经验证的报告人 构建体将在动物生产之前在三个安全港位点之一整合到猪基因组中 体细胞核移植（克隆）。这些创始动物及其后代的报告活性将被 其特征在于全身成像和单细胞分析。经验证的报告者、相关数据和 然后将动物模型提供给体细胞基因编辑联盟进行分发。这些新颖 模型将使新的发现能够表征治疗递送、DNA修复偏好和脱靶 对体内任何组织或细胞类型的风险。 0

PROJECT SUMMARY Precise gene inactivation, gene addition, and gene repair in patient cells are all part of a new paradigm in gene therapy. However, the full potential of these gene editing therapies can only be realized when it becomes possible to treat each tissue directly in the body of the patient. This goal requires novel gene editing delivery vehicles and routes of administration where uptake kinetics, penetration, and distribution are well established. A first step to achieve this goal is to develop model animals that mimic size, physiology, and aging of human patients. Swine models meet these criteria to enable in utero, pediatric, and adult gene editing therapies. Accordingly, Recombinetics has been using gene editing to create a suite of swine models with humanized disease alleles for the past 6 years to augment preclinical research and development. While these models are valued for efficacy studies, findings from these animals are not broadly applicable for optimization in vivo editing. To address this limitation, we propose to develop a suite of swine models and vectors capable of reporting gene-editing outcomes with a combination of in vivo (whole animal) and single cell readouts. Our modular design and production pipeline will enable rapid and reliable modeling of most human disease alleles within a single reporter system. The proposed models will report outcomes of two types of gene editing tools: 1) those that cut or nick DNA to stimulate DNA repair, ie. CRISPR/Cas9, TALENs, ZFNs, and 2) base-editors that convert selected DNA bases to another base at the target site without creating a double strand break. The validated reporter constructs will be integrated into the swine genome at one of three safe-harbor loci prior to animal production by somatic cell nuclear transfer (cloning). Reporter activity in these founder animals and their offspring will be characterized by whole body imaging and single cell analysis. The validated reporters, associated data, and animal models will then be provided to the Somatic Cell Gene Editing consortium for distribution. These novel models will enable new discoveries to characterize therapeutic delivery, DNA repair preferences, and off-target risk for any tissue or cell type in the body. 0