Targeted Integration of a Lentiviral Vector

项目概要：Sinn博士的长期目标是从事一项重大研究的学术事业， 研究生物学和慢病毒载体的发展。这将通过以下方式实现： Drs.麦克雷和Voytas和部门的支持。爱荷华州大学的儿科教授。立即 Sinn博士感兴趣的是慢病毒载体在广泛的基因治疗中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， 和消化的表现，是直接适用于使命的NIDDK。持续表达a 为了获得治疗性转基因，逆转录病毒载体必须整合到宿主细胞染色体中。这一关键过程 使慢病毒载体成为实现终身基因递送的有吸引力的工具;然而， 逆转录病毒的整合存在固有的危险和基因表达的变异。如果融合可以 限制于优选的基因组位点，慢病毒载体的安全性和实用性将大大提高。在 该建议是一种新的策略，研究修饰整合酶（IN）基因的猫免疫缺陷 病毒（FIV）为基础的慢病毒载体，以实现限制性整合。提出了一种“网络共享”模型， 含有蛋白质结合结构域和DNA结合结构域的工程化杂合蛋白将指导 将慢病毒载体整合复合物结合到染色体DNA上的优选位点。提出了三个目标： 1)确定FIV IN可被修改并仍保留其功能的容量，由 滴定测定以及体外催化和整合酶测定; 2）证实高亲和力蛋白-蛋白 通过酵母双杂交试验研究了修饰的FIV IN与拴系蛋白的相互作用，并证明， 这些复合物可以介导细胞系统中的限制性整合。这些研究将集中在最终的 目的是，3）将修饰的载体的整合位点定位到基因组DNA中，并证明限制性的整合位点。 在其中系链蛋白共表达的那些细胞中的整合模式。 相关性：慢病毒载体具有持续纠正遗传疾病的潜力。但这种 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。成功地将位点限制性慢病毒载体整合到人基因组中 DNA将在基因治疗领域有令人兴奋和广泛的应用。

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.