RECOMBINASE MEDIATED SITE SPECIFIC INTEGRATION FOR GENE

描述（改编自应用）缺乏持久性是当前基因治疗载体的主要问题。虽然逆转录病毒载体通过整合到染色体中获得持久性，但这些载体随机整合，导致突变和基因表达的可预测性丧失。大多数其他向量不能有效地集成，并随着时间的推移而丢失。我们希望开发一种方法，提供永久基因治疗的染色体整合感兴趣的基因在一个位点特异性和有效的方式。为了提供这些属性，我们正在使用一种重组酶，它可以在自己的识别位点上以高效率实现精确的重组。特别是，我们正在使用CRE重组。整合只会发生在哺乳动物基因组中预先选择的位点上，这些位点与天然LOX位点有足够的同源性，可以被CRE酶有效地用于重组。我们将通过计算机在基因组数据库中找到这些伪LOX位点，然后开发快速检测方法来评估它们的重组频率。在细菌和人类细胞的快速测定中，那些介导有效重组的位点将被分析为位点特异性整合到染色体中。将开发一种优化的CRE表达系统，以整合和最小化切除。该试验系统将在人类和小鼠细胞染色体中定义的、表达良好的位点上实现高效、永久、位点特异性的整合。该系统可与病毒或非病毒基因传递方法结合使用。接下来的实验将使用这种方法在老鼠身上测试整合，为在病人身上测试做准备。

DESCRIPTION (adapted from the application) Lack of permanence is a major problem with current gene therapy vectors. While retrovirus vectors attain permanence by integrating into chromosomes, these vectors integrate randomly, leading to mutagenesis and loss of predictability of gene expression. Most other vectors fail to integrate efficiently and are lost over time. We wish to develop a method to provide permanent gene therapy by chromosomal integration of the gene of interest in a site-specific and efficient manner. To supply these attributes, we are using a recombinase enzyme that can achieve precise recombination at a high efficiency, restricted to its own recognition site. In particular, we are using the CRE recombines. Integration will occur only at pre-chosen sites in mammalian genomes that have sufficient homology to the native LOX sites to be used efficiently in recombination by the CRE enzyme. We will find such pseudo LOX sites in the genome database by computer, then develop rapid assays to evaluate their recombination frequencies. Those sites mediating efficient recombination in rapid assays in bacteria and human cells will be analyzed for site-specific integration into the chromosomes. A CRE expression system optimized for integration, and minimizing excision, will be developed. The pilot system will achieve efficient, permanent, site-specific integration at defined, well- expressed sites in the chromosomes of human and mouse cells. This system can be used in combination with viral or non-viral gene delivery methods. The next experiments will use such methods to test integration in mice, preparatory to testing in patients.