RECOMBINASE MEDIATED SITE SPECIFIC INTEGRATION FOR GENE

描述（改编自本申请）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永久性，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。大多数其他载体不能有效地整合，并随着时间的推移而丢失。我们希望开发一种方法，通过以位点特异性和有效的方式将感兴趣的基因整合到染色体上来提供永久的基因治疗。为了提供这些属性，我们正在使用一种重组酶，该酶可以以高效率实现精确重组，仅限于其自身的识别位点。特别是，我们正在使用CRE重组。整合将仅发生在哺乳动物基因组中与天然LOX位点具有足够同源性的预选位点处，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假LOX位点，然后开发快速检测方法来评估它们的重组频率。将分析在细菌和人细胞中快速测定中介导有效重组的那些位点的位点特异性整合到染色体中。一个CRE表达系统优化整合，并尽量减少切除，将开发。中试系统将在人和小鼠细胞染色体中定义的良好表达位点实现有效、永久、位点特异性整合。该系统可以与病毒或非病毒基因递送方法组合使用。接下来的实验将使用这种方法来测试小鼠的整合，为患者的测试做准备。

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.