Targeted Integration of a Lentiviral Vector

慢病毒载体的靶向整合

• 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
• 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

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一项重大研究中从事学术职业 大学研究慢病毒载体的生物学和发展。这将在 博士。麦克雷和沃伊塔斯以及爱荷华大学儿科系的支持。直接 Sinn博士的兴趣是慢病毒载体的一般安全和实用性，用于广泛的基因 治疗应用，例如囊性纤维化。囊性纤维化的基因治疗，一种肺部的疾病 消化表现形式直接适用于NIDDK的任务。持续表达 治疗转基因，逆转录病毒载体必须集成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身基因递送的有吸引力的工具；但是，非特异性的性质 逆转录病毒整合表达了基因表达的固有危害和变化。是否可以集成 仅限于首选的基因组基因局，慢病毒载体的安全性和效用将得到极大的改善。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，以实现限制整合。提出了一个“束缚”模型 包含蛋白质结合结构域和DNA结合结构域的工程混合蛋白将导演 在染色体DNA上的首选位点的慢病毒载体积分复合物。提出了三个目标： 1）确定可以修改FIV的能力并仍保留其功能，如 滴定测定以及体外催化和整合酶测定； 2）确认高亲和力蛋白质蛋白质 通过酵母双杂交测定法，改性FIV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限制整合。这些研究将集中于最终 3）将修饰载体的集成位点映射到基因组DNA中，并证明了受限制的 在那些共同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。但是，这样 向量将非专门集成到宿主基因组中，因此存在破坏正常基因的风险 在插入部位的功能。成功地限制了慢病毒矢量整合到人类基因组中 DNA在基因治疗领域中会有令人兴奋的广泛应用。