Next generation transposon vectors for genome engineering

非病毒基因传递系统受到其活性和靶向整合能力的限制。高效和 有针对性地将DNA整合到哺乳动物和人类基因组中仍然是一项重大挑战，并取得了成功 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传递系统，是一种剪切粘贴的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年了。我们 已经在第一个基础上重新设计了PB-转座体(带有转座子DNA的转座酶)- 我们最近与合作者一起发表的PB转座体的三维结构 Fred Dyda博士(Chen等人，《自然通讯》，2020)。我们的下一代PB转座子 (NgPB)显示出比以前可以实现的更大的活动和定向整合的潜力。在……里面 具体目标1，我们将为人类细胞基因组工程设计和测试ngPB。我们将评估 每个人类细胞的整合位点概况和转座子整合的拷贝数。我们将修改主服务器 人T细胞活体表达并检测其细胞治疗能力，我们将启用转座酶蛋白的转染。 在具体目标2中，我们将在体内设计和测试ngPB用于基因传递。我们将评估基因传递 报告和治疗性转基因到小鼠肝脏，测试转基因小鼠的发育效率，以及 评价杂交腺相关病毒(AAV)-ngPB介导的难以到达器官的基因传递。在……里面 具体目标3，我们将设计和测试在人类细胞中靶向整合的ngPB。我们还将绘制 已知的PB的蛋白质-蛋白质相互作用结构域影响其在人细胞中的靶点选择和测试PB 蛋白质修饰，以允许更大的灵活性来操纵PB基因组靶点选择。这个 拟议的研究将对基因组工程产生变革，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.