Transferring integrase technology to animals

描述（由申请人提供）：我们开发了新技术， 哺乳动物细胞中的位点特异性整合。该技术涉及使用 介导高效整合的新型噬菌体整合酶 识别位点。酶识别的位点出现的频率很低， 哺乳动物基因组，并可用于靶向整合。我们已经证实了这一点 技术在哺乳动物组织培养细胞，它是准备转移到 动物体内实验现在对于应用 技术，以改善现有的基因治疗方法， 构建转基因动物。 拟议的实验包括产生转基因小鼠， 整合酶的识别位点。这些动物将用于基因 治疗研究和改善基础技术，创造转基因 哺乳动物我们将在野生型，转基因， 通过流体动力学方法将质粒DNA导入肝脏， 尾静脉注射法。我们将携带基因的质粒 噬菌体整合酶沿着与携带治疗基因的质粒和 整合酶的识别位点。这种酶将介导位点特异性 将治疗性基因整合到基因组中的由免疫球蛋白识别的位点处， 酵素我们将利用这项技术开发有效的永久性基因疗法 通过引入α 1-抗胰蛋白酶治疗肺气肿和血友病B， 因子IX基因。这些实验的成功将导致这一应用 创新技术应用于其他基因治疗环境， 在病人身上进行临床试验。

DESCRIPTION (provide by applicant): We have developed new technology for site-specific integration in mammalian cells. The technology involves use of novel phage integrases that mediate efficient integration at compact recognition sites. Sites recognized by the enzymes occur at low frequency in mammalian genomes and can be used to target integration. We have validated this technology in mammalian tissue culture cells, and it is ready for transfer to animals. In vivo experiments are now essential for application of the technology to improve existing methodologies for gene therapy and for construction of transgenic animals. The proposed experiments include the generation of transgenic mice that carry recognition sites for the integrases. These animals will be used for gene therapy studies and to improve basic technology for creation of transgenic mammals. We will carry out gene therapy studies in wild-type, transgenic, and disease model mice by introducing plasmid DNA to the liver by the hydrodynamic tail vein injection method. We will deliver a plasmid carrying the gene for a phage integrase along with a plasmid carrying the therapeutic gene and a recognition site for the integrase. The enzyme will mediate site-specific integration of the therapeutic gene into the genome at sites recognized by the enzyme. We will use this technology to develop effective permanent gene therapy treatments for emphysema and hemophilia B by introducing the a1-antitrypsin and factor IX genes. Success in these experiments will lead to application of this innovative technology to other gene therapy settings and progress toward clinical trials in patients.