Transferring Integrase Technology to Animals

描述（由申请人提供）：本提案的目标是通过动物研究继续推进这种创新的位点特异性整合技术在基因治疗中的应用。该技术使用噬菌体phiC31整合酶，在基因组的首选位置提供安全有效的基因治疗载体特异性位点整合。phic31介导的整合提供了强大的、长期的整合治疗基因表达，没有随机整合的风险。在两年的资助期间，我们证明了phiC31整合酶技术在小鼠肝脏中长期、高水平地表达因子IX方面是有效的。我们还证实了移植到小鼠模型上的人皮肤中VII胶原和层粘连蛋白B3的治疗性表达，以及人α 1抗胰蛋白酶在小鼠肝脏中的表达。此外，我们还利用这项技术创造了转基因动物。

DESCRIPTION (provided by applicant): The goal of this proposal is to continue advancing this innovative site-specific integration technology toward utility in gene therapy through studies in animals. The technology uses the phage phiC31 integrase to provide safe and efficient site-specific integration of incoming gene therapy vectors at preferred locations in the genome. PhiC31-mediated integration provides robust, long-term expression of the integrated therapeutic gene, without the risk of random integration. During the two years of the grant to date, we demonstrated that the phiC31 integrase technology was effective in providing long-term, high level liver expression of factor IX in mice. We also demonstrated therapeutic expression of collagen VII and laminin B3 in human skin grafted on to mouse models and expression of human alpha1 antitrypsin in mouse liver. In addition, we used the technology to create transgenic animals. We now wish to continue development of the integrase technology for long-term gene therapy by moving to larger animal models in two important tissues, liver and muscle. We have already demonstrated effective DNA delivery methods for these tissues. In both liver and muscle we observed more robust and longer-lived gene expression when phiC31 integrase technology was used, compared to unintegrated plasmid DNA. We will employ hydrodynamic delivery of the phiC31 integrase system to express therapeutic levels of factor IX from the livers of rats, rabbits, and dogs. We will also explore alternative methods to deliver the integrase system to liver. In addition, we will use the phiC31 integrase technology, delivered to muscle by DNA injection and electroporation, for integration and long-term expression of plasmids bearing VEGF and other angiogenic factors to correct ischemia in mice, rats, and rabbits. By scaling up to animals more similar to humans, these experiments will move the site-specific integrase technology closer to the clinic.