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：
10688194
负责人：
MATTHEW H WILSON
金额：
$ 49.15万
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/10688194
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 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.

非病毒基因输送系统受其活性和靶向整合能力的限制。高效和将DNA的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及其成功将对生物技术和治疗应用产生广泛的影响。 Piggybac（PB）转座子系统是最活跃的非病毒基因递送系统，是一种切割和paste的DNA转座子已用于哺乳动物和人类细胞的基因组工程已有15年以上。我们已经重新设计了基于第一我们最近与合作者一起发布的PB转座体的三维结构弗雷德·迪达（Fred Dyda）博士（Chen等人，《自然通讯》，2020年）。我们的下一代PB转座体（NGPB）比以前可实现的靶向整合表现出更大的活性和潜力。在具体目标1，我们将设计和测试NGPB的人类细胞基因组工程。我们将评估每个人类细胞的集成位点轮廓和副本集成的副盆地积分。我们将修改主人类T细胞外生物并测试其细胞疗法的能力，我们将启用转座蛋白转染。在特定的目标2中，我们将在体内设计和测试NGPB的基因输送。我们将评估基因输送记者和治疗性转基因对小鼠肝脏，转基因小鼠发育效率的测试，以及评估杂化腺相关病毒（AAV）-NGPB介导的基因输送到难以到达器官。在特定的目标3，我们将设计和测试NGPB在人类细胞中的靶向整合。 We will also map the protein-protein interaction domain of PB known to affect its target site selection in human cells and test PB 蛋白质修饰以使操纵PB基因组靶位位点选择方面更大。这拟议的研究将是基因组工程的变革性，并对生物技术产生广泛的影响和治疗应用。