SITE-SPECIFIC INTEGRATION FOR GENE THERAPY

这项工作的目标是创建一个系统， 基因治疗载体位点特异性整合到染色体中。这 目前，在所有情况下都缺乏这种功能。这里开发的创新系统 将提供引入基因的长期表达， 靶细胞这种结果在大多数基因治疗策略中是理想的。在 为了实现有效和特定地点的整合，Calos博士将使用 在哺乳动物细胞中工作的原核重组酶， 辅因子这些酶具有长的识别位点， 发生在哺乳动物的基因组中。然而，首席研究员已经表明， 酶也识别少量的天然序列，称为假位点， 与天然识别位点同源。这些伪站点可以用于 用于位点特异性整合到哺乳动物染色体中。校长 研究者将使用以下方法定位三种重组酶的这些位点： 整合效率和在染色体环境中良好的基因表达。 将对重组酶进行蛋白质工程改造， 效率和改变DNA识别特异性。基因治疗实验将 通过使用引入，用最佳酶/假位点候选物启动 质粒DNA。这里开发的新颖的特定地点整合策略可以 用于为大多数当前的基因治疗载体增加价值。

The goal of this work is to create a system that will produce site-specific integration of gene therapy vectors into the chromosomes. This feature is currently lacking in all cases. The innovative system developed here will provide long-term expression of introduced genes, for the lifetime of the target cells. This outcome is desirable in most gene therapy strategies. In order to achieve efficient and site-specific integration, Dr. Calos will use prokaryotic recombinase enzymes that work in mammalian cells with no added co-factors. These enzymes have long recognition sites that are not expected to occur in mammalian genomes. However, the principal investigator has shown that enzymes also recognize a small number of native sequences, called pseudo sites, with homology to the native recognition site. These pseudo sites can be used for site-specific integration into mammalian chromosomes. The principal investigator will locate such sites for three recombinase enzymes, using integration efficiency and good gene expression in a chromosomal context. Protein engineering will be carried out on the recombinases to improve efficiency and alter DNA recognition specificity. Gene therapy experiments will be initiated with the best enzyme/pseudo site candidates by using introduction of plasmid DNA. The novel site-specific integration strategy developed here can be used to add value to most current gene therapy vectors.