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Next generation transposon vectors for genome engineering

用于基因组工程的下一代转座子载体

基本信息

批准号：
10501335
负责人：
MATTHEW H WILSON
金额：
$ 52.55万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10501335
关键词：
Advanced Development Affect Animal Model Biological Assay Biotechnology Cell Line Cell Therapy Cells Clinical Trials Communication Complex DNA DNA Integration DNA Transposons Development Embryo Engineering Enzymes Gene Delivery Gene Transfer Genome Genome engineering Genomics Human Human Engineering Human Genome Hybrids In Vitro Injections Inverted Terminal Repeat Kidney Liver Mediating Methodology Modification Mus Nature Oncogenes Organ Paste substance Plasmids Post-Translational Protein Processing Production Protein Binding Domain Protein-Protein Interaction Map Proteins Publishing Recombinant Proteins Reporter Site System T-Lymphocyte Testing Therapeutic Transfection Transgenic Animals Transgenic Mice Transposase Viral base clinically relevant cost flexibility functional genomics gene delivery system gene discovery gene therapy genotoxicity immunogenicity in vivo induced pluripotent stem cell integration site mammalian genome next generation next generation sequencing non-viral gene delivery novel strategies repaired stable cell line success therapeutic transgene three dimensional structure transgene expression vector genome

项目摘要

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.

非病毒基因传递系统受到其活性和靶向整合能力的限制。高效且将 DNA 有针对性地整合到哺乳动物和人类基因组中仍然是一个重大挑战及其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 DNA 转座子，用于哺乳动物和人类细胞的基因组工程已超过 15 年。我们基于第一个-重新设计了PB-转座体（带有转座子DNA的转座酶）我们最近与合作者发表的 PB 转座体的三维结构 Fred Dyda 博士（Chen 等人，Nature Communications，2020）。我们的下一代 PB 转座体（ngPB）展示了比以前可实现的更大的活动和有针对性的整合潜力。在具体目标1，我们将设计和测试ngPB用于人类细胞的基因组工程。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。我们将修改初级人类 T 细胞离体并测试其细胞治疗能力，我们将实现转座酶蛋白转染。在具体目标 2 中，我们将设计和测试 ngPB 在体内的基因传递。我们将评估基因传递对小鼠肝脏进行报告基因和治疗性转基因，测试转基因小鼠发育的效率，以及评估杂交腺相关病毒 (AAV)-ngPB 介导的基因递送至难以到达的器官。在具体目标 3，我们将设计和测试 ngPB 以在人体细胞中进行靶向整合。我们还将绘制地图 PB 的蛋白质-蛋白质相互作用结构域已知影响其在人类细胞中的靶位点选择并测试 PB 蛋白质修饰可以更灵活地操纵 PB 基因组靶位点选择。这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。