Next generation transposon vectors for genome engineering

非病毒基因递送系统受到其活性和靶向整合能力的限制。高效和 将DNA靶向整合到哺乳动物和人类基因组中仍然是一个重大挑战， 将对生物技术和治疗应用产生广泛影响。piggyBac（PB）转座子系统 是最活跃的整合非病毒基因递送系统，并且是剪切和粘贴DNA转座子， 已经用于哺乳动物和人类细胞的基因组工程超过15年。我们 基于第一个- 我们最近与合作者发表的PB转座体的三维结构 博士Fred Dyda（Chen等人，Nature Communications，2020）。我们的下一代PB转座体 （ngPB）显示出比以前可实现的更大的活性和有针对性的整合潜力。在 具体目标1，我们将工程化和测试ngPB用于人类细胞的基因组工程。我们将评估 整合位点概况和每个人细胞转座子整合拷贝数。我们将修改主要 体外培养人T细胞并测试其细胞治疗能力，我们将使转座酶蛋白转染成为可能。 在具体目标2中，我们将设计和测试ngPB用于体内基因递送。我们将评估基因传递， 报告基因和治疗性转基因至小鼠肝脏，测试转基因小鼠发育的效率，和 评估杂合腺相关病毒（AAV）-ngPB介导的基因递送至难以到达的器官。在 具体目标3，我们将工程化并测试ngPB在人细胞中的靶向整合。我们还将绘制 已知影响其在人细胞中的靶位点选择的PB的蛋白质-蛋白质相互作用结构域和测试PB 蛋白质修饰以允许在操纵PB基因组靶位点选择中具有更大的灵活性。的 拟议的研究将对基因组工程产生变革性影响，并对生物技术产生广泛影响。 和治疗应用。

Non-viral gene delivery systems are limited by their activity and targeted integration capability. Efficient and targeted integration of DNA into mammalian and human genomes remains a major challenge and its success would have wide impact for biotechnology and therapeutic applications. The piggyBac (PB) transposon system is the most active integrating non-viral gene delivery system and is a cut-and-paste DNA transposon that has been used for genome engineering of mammalian and human cells for more than 15 years. We have re-engineered the PB-transpososome (transposase with transposon DNA) based on the first- ever three-dimensional structure of the PB transpososome that we recently published with our collaborator Dr. Fred Dyda (Chen et al., Nature Communications, 2020). Our next-generation PB transpososome (ngPB) demonstrates greater activity and potential for targeted integration than was previously achievable. In specific aim 1, we will engineer and test ngPB for genome engineering of human cells. We will evaluate the integration site profile and copy number of transposon integrations per human cell. We will modify primary human T cells ex vivio and test their ability for cell therapy, and we will enable transposase protein transfection. In specific aim 2, we will engineer and test ngPB for gene delivery in vivo. We will evaluate gene delivery of reporter and therapeutic transgenes to mouse liver, test for efficiency in development of transgenic mice, and evaluate hybrid adeno-associated viral (AAV)-ngPB mediated gene delivery to difficult to reach organs. In specific aim 3, we will engineer and test ngPB for targeted integration in human cells. We will also map the protein-protein interaction domain of PB known to affect its target site selection in human cells and test PB protein modifications to allow greater flexibility in manipulating PB genomic target site selection. The proposed studies will be transformative for genome engineering and have broad impact for biotechnology and therapeutic applications.