Targeted Integration of a Lentiviral Vector

慢病毒载体的靶向整合

• 批准号: 7170056
• 负责人: PATRICK L SINN
• 金额: $ 11.7万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 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/7170056
• 关键词: Affinity; Binding; Biological Assay; Biology; Cell Line; Cell-Free System; Cells; Chimeric Proteins; Chromosomes; Complementary DNA; Complex; Cystic Fibrosis; DNA; DNA Binding Domain; Development; Disease; Engineering; Escherichia coli; Family Felidae; Feline Immunodeficiency Virus; Figs - dietary; Gene Delivery; Gene Expression; Gene Transfer; Genes; Genome; Genomics; Goals; Hereditary Disease; Human; Immunologic Deficiency Syndromes; In Vitro; Insertional Mutagenesis; Integrase; Iowa; Lentivirus Vector; Life; Link; Lung; Lung diseases; Mammalian Cell; Maps; Mediating; Mission; Modeling; National Institute of Diabetes and Digestive and Kidney Diseases; Nature; Oligonucleotides; Pattern; Pediatrics; Peptides; Polymerase Chain Reaction; Process; Production; Proline; Protein Binding; Protein Binding Domain; Proteins; RNA; Range; Research; Retroviral Vector; Risk; Safety; Site; Southern Blotting; Standards of Weights and Measures; System; Tandem Repeat Sequences; Two-Hybrid System Techniques; Universities; Variant; Virus; Virus Integration; Yeasts; base; career; gene function; gene therapy; hazard; hybrid protein; improved; interest; novel strategies; prevent; protein aminoacid sequence; protein protein interaction; research study; therapeutic transgene; tool; vector; yeast protein

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.

项目概述：辛恩博士的长期目标是在一项主要研究中追求学术生涯 大学研究慢病毒载体的生物学和发展。这将在以下方面实现 McCray博士和Voytas博士以及国防部的支持。爱荷华大学的儿科博士。最直接的 辛恩博士感兴趣的是用于广泛基因范围的慢病毒载体的一般安全性和实用性。 治疗应用，如囊性纤维化。囊性纤维化的基因治疗，这是一种有肺部疾病 和消化表现，是直接适用于NIDDK的使命。坚持不懈地表达 为了治疗转基因，逆转录病毒载体必须整合到宿主细胞的染色体中。这一关键过程 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 逆转录病毒整合带来了固有的危险和基因表达的变异。如果整合可以是 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得到极大的提高。在……里面 本研究提出了一种修饰猫免疫缺陷整合酶(IN)基因的新策略 基于病毒(FIV)的慢病毒载体实现限制性整合。提出了一种“系绳”模型，其中 一种含有蛋白质结合域和DNA结合域的工程杂交蛋白将定向 慢病毒载体整合到染色体DNA上的首选位置。提出了三个目标： 1)确定FIV IN可以修改到的容量，并仍保留其功能，由以下方式确定 滴定试验、体外催化试验和整合酶试验；2)确认高亲和力蛋白质 酵母双杂交实验证明修饰后的FIV IN与系留蛋白的相互作用 这些复合体可以调节细胞系统中的限制性整合。这些研究将集中于最终的 目的：3)将修饰载体的整合位点定位到基因组DNA中，并证明 在那些共同表达系留蛋白的细胞中的整合模式。 相关性：慢病毒载体具有持久纠正遗传病的潜力。然而，这样的 载体非特异性地整合到宿主基因组中，因此存在破坏正常基因的风险 在插入部位起作用。位点限制性慢病毒载体成功整合到人类基因组中 DNA在基因治疗领域将有令人兴奋和广泛的应用。