Targeted Integration of a Lentiviral Vector

慢病毒载体的靶向整合

基本信息

批准号：
7337315
负责人：
PATRICK L SINN
金额：
$ 11.97万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-01 至 2009-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7337315
关键词：
Affinity Binding Biological Assay Cell Line Cell-Free System Cells Chimeric Proteins Chromosomes Complementary DNA Complex Cystic Fibrosis DNA DNA Binding Domain Engineering Escherichia coli Feline Immunodeficiency Virus Figs - dietary Gene Delivery Gene Expression Gene Transfer Genome Genomics Human In Vitro Insertional Mutagenesis Integrase Lentivirus Vector Life Link Lung diseases Mammalian Cell Maps Mediating Nature Oligonucleotides Peptides Polymerase Chain Reaction Principal Investigator Process Production Proline Protein Binding Protein Binding Domain Proteins RNA Research Retroviral Vector Site Southern Blotting Standards of Weights and Measures Tandem Repeat Sequences Two-Hybrid System Techniques Variant Yeasts base hazard hybrid protein novel strategies prevent programs protein aminoacid sequence protein protein interaction research study therapeutic transgene tool vector

项目摘要

Project Summary: The long-term objective of Dr. Sinn is to pursue an academic career at a major research university studying the biology and development of lentiviral vectors. This will be achieved with the aid of Drs. McCray and Voytas and the support of the Dept. of Pediatrics at the University of Iowa. The immediate interest of Dr. Sinn is the general safety and utility of lentiviral vectors for use in a broad range of gene therapy applications, such as cystic fibrosis. Gene therapy for cystic fibrosis, a disease that has pulmonary and digestive manifestations, is directly applicable to the mission of the NIDDK. To persistently expressa therapeutic transgene, a retroviral vector must integrate into a host cell chromosome. This critical process makes lentiviral vectors an attractive tool to achieve life-long gene delivery; however, the nonspecific nature of retroviral integration presents inherent hazards and variations in gene expression. If integration could be restricted to preferred genomic loci, the safety and utility of lentiviral vectors would be vastly improved. In this proposal a novel strategy is investigated to modify the integrase (IN) gene of a feline immunodeficiency virus (FIV)-based lentiviral vector to achieve restricted integration. A "tethering" model is proposed in which an engineered hybrid protein that contains a protein binding domain and a DNA binding domain will direct the lentiviral vector integration complex to preferred sites on chromosomal DNA. Three aims are proposed: 1) determine the capacity to which FIV IN can be modified and still retain its function, as determined by titering assays as well as in vitro catalytic and integrase assays; 2) confirm high affinity protein-protein interactions of modified FIV IN to the tethering protein by yeast two-hybrid assays and demonstrate that these complexes can mediate restricted integration in a cell-system. These studies will focus the ultimate goal to, 3) map integration sites of the modified vector into genomic DNA and demonstrate a restricted pattern of integration in those cells in which the tethering protein is co-expressed. Relevance: Lentiviral vectors have the potential to persistently correct genetic diseases. However, such vectors integrate nonspecifically into the host genome and therefore present a risk of disrupting normal gene function at the site of insertion. Successful site-restricted lentiviral vector integration into human genomic DNA would have exciting and broad applications in the gene therapy field.

项目摘要：Sinn 博士的长期目标是在主要研究领域追求学术生涯大学研究慢病毒载体的生物学和开发。这将在以下方面实现：博士。麦克雷和沃伊塔斯以及爱荷华大学儿科系的支持。即时的 Sinn 博士感兴趣的是慢病毒载体在广泛的基因应用中的总体安全性和实用性治疗应用，例如囊性纤维化。囊性纤维化（一种肺部疾病）的基因治疗和消化表现，直接适用于 NIDDK 的使命。坚持不懈地表达治疗性转基因，逆转录病毒载体必须整合到宿主细胞染色体中。这个关键过程使慢病毒载体成为实现终生基因传递的有吸引力的工具；然而，非特异性逆转录病毒整合存在固有的危险和基因表达的变化。如果整合可以限制在优选的基因组位点，慢病毒载体的安全性和实用性将大大提高。在该提案研究了一种新的策略来修改猫免疫缺陷的整合酶（IN）基因基于病毒（FIV）的慢病毒载体实现限制性整合。提出了“束缚”模型，其中包含蛋白质结合结构域和 DNA 结合结构域的工程杂合蛋白将指导将慢病毒载体整合复合物整合到染色体 DNA 上的首选位点。提出了三个目标： 1) 确定 FIV IN 可以修改并仍然保留其功能的容量，由下式确定滴定测定以及体外催化和整合酶测定； 2) 确认蛋白-蛋白高亲和力通过酵母双杂交测定修饰的 FIV IN 与束缚蛋白的相互作用，并证明这些复合物可以介导细胞系统中的有限整合。这些研究将集中于最终目标是，3) 将修饰载体的整合位点映射到基因组 DNA 中，并证明限制性共表达束缚蛋白的细胞中的整合模式。相关性：慢病毒载体具有持续纠正遗传疾病的潜力。然而，这样的载体非特异性整合到宿主基因组中，因此存在破坏正常基因的风险在插入位点发挥作用。成功将位点限制性慢病毒载体整合到人类基因组中 DNA 在基因治疗领域将具有令人兴奋且广泛的应用。