Transferring Integrase Technology to Animals

描述(由申请人提供)：这项提案的目标是通过动物实验继续推进这一创新的特定部位整合技术，以实现在基因治疗中的实用。这项技术使用噬菌体phiC31整合酶来提供安全和高效的输入基因治疗载体在基因组中首选位置的定点整合。PhiC31介导的整合提供了整合的治疗基因的强大的、长期的表达，而没有随机整合的风险。到目前为止，在拨款的两年中，我们证明了phiC31整合酶技术在小鼠肝脏提供长期、高水平的因子IX表达是有效的。我们还证明了在移植到小鼠模型上的人皮肤中具有治疗性的VII型胶原和层粘连蛋白B3的表达，以及在小鼠肝脏中表达人α1抗胰蛋白酶。此外，我们还利用这项技术创造了转基因动物。 我们现在希望通过转移到两个重要组织-肝脏和肌肉的更大动物模型来继续开发用于长期基因治疗的整合酶技术。我们已经为这些组织展示了有效的DNA输送方法。在肝脏和肌肉中，我们观察到，与未整合的质粒DNA相比，使用phiC31整合酶技术时，肝脏和肌肉中的基因表达更强劲、寿命更长。我们将使用流体动力学传递phiC31整合酶系统来表达来自大鼠、兔和狗肝脏的治疗水平的因子IX。我们还将探索将整合酶系统输送到肝脏的替代方法。此外，我们将使用phiC31整合酶技术，通过DNA注射和电穿孔传递到肌肉，整合和长期表达携带血管内皮生长因子和其他血管生成因子的质粒，以纠正小鼠、大鼠和兔的缺血。通过扩大到更接近人类的动物，这些实验将使定点整合酶技术更接近临床。

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.