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

项目总结 患者细胞中精确的基因失活、基因添加和基因修复都是新范式的一部分。 在基因治疗方面。然而，这些基因编辑疗法的全部潜力只有当它 使直接在患者体内治疗每个组织成为可能。这一目标需要新的基因编辑 吸收动力学、渗透性和分布性良好的给药载体和给药途径 已经成立了。实现这一目标的第一步是开发模拟大小、生理和 人类患者的老龄化。猪模型符合这些标准，可以在子宫、儿科和成人体内进行基因编辑 治疗。因此，Recombinetics一直在使用基因编辑来创建一套猪模型 过去6年人源化疾病等位基因，以加强临床前研究和开发。而这些 模型在疗效研究中很有价值，但这些动物的研究结果并不广泛适用于优化 活体编辑。为了解决这一局限性，我们建议开发一套能够 使用体内(整个动物)和单细胞读数相结合的方法报告基因编辑结果。我们的 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可靠建模成为可能 在单一的记者系统内。 建议的模型将报告两种类型的基因编辑工具的结果：1)那些切割或划破的工具 刺激DNA修复的DNA，即。CRISPR/CAS9、TALEN、ZFN和2)用于转换选定内容的基本编辑器 DNA碱基连接到目标位置的另一个碱基，而不会造成双链断裂。经验证的记者 在动物生产之前，构建物将整合到猪的基因组中的三个安全港位置之一。 通过体细胞核移植(克隆)。记者在这些创始动物及其后代中的活动将是 以全身成像和单细胞分析为特征。经过验证的记者、关联数据和 动物模型随后将被提供给体细胞基因编辑联盟进行分发。这些小说 模型将使新的发现能够表征治疗提供、DNA修复偏好和脱靶 对体内任何组织或细胞类型都有风险。 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