SITE-SPECIFIC INTEGRATION FOR GENE THERAPY

这项工作的目标是创建一种将产生 基因治疗载体与染色体的定点整合。这 目前所有情况下都缺少此功能。这里开发的创新系统 将提供导入基因的长期表达，在 目标单元格。这一结果在大多数基因治疗策略中都是可取的。在……里面 为了实现高效和特定地点的整合，卡洛斯博士将使用 在哺乳动物细胞中不添加任何添加剂的原核重组酶 共同因素。这些酶有很长的识别位点，而不是预期的 存在于哺乳动物的基因组中。然而，首席调查员已经表明， 酶还识别少量的自然序列，称为伪位点， 与本地识别位点有同源性。这些伪站点可以用来 定位整合到哺乳动物的染色体中。校长 研究人员将使用以下方法确定三种重组酶的这样的位置 在染色体背景下的整合效率和良好的基因表达。 将对重组酶进行蛋白质工程以提高 效率和改变DNA识别的特异性。基因治疗实验将 用最好的酶/伪位点通过使用介绍来启动 质粒DNA的表达。这里开发的新的特定于站点的集成策略可以 用于增加目前大多数基因治疗载体的价值。

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.