Retinal Gene Therapy by Site-Specific Integration

描述（由申请人提供）：一项强大的基因治疗新技术现已问世。该技术使用来自噬菌体phiC31的整合酶，介导治疗基因的染色体整合到哺乳动物基因组中有限数量的天然序列中。这种创新的技术是唯一一种提供有效的位点特异性染色体整合，而不是随机的。该方法还提供了治疗基因的永久、强表达。整合酶系统已在许多组织、物种和疾病的动物模型中得到验证。本提案的重点是使用phiC31整合酶系统来创建一种安全、简单和永久地将基因转移到大型哺乳动物视网膜的新方法。我们展示了一种电穿孔方法，可以有效地将DNA传递到大鼠视网膜，并表明整合酶在视网膜色素上皮中提供了一个标记基因的强、长期表达。现在将建立方法来实现有效的基因传递和位点特异性整合到兔子的视网膜中，作为后来狗和人类工作的模型。创新的电穿孔方法将与物理学和眼科学的合作者一起开发，以实现手术电极的放置，以提供有效和精确的治疗性DNA递送到RPE，同时将组织损伤降到最低。phiC31整合酶将用于提供位点特异性基因组整合和视网膜中转移基因的长期表达。体外模型系统将首先优化给药参数，而兔眼荧光素酶成像将用于长期的体内研究。为了为RPE65犬的后续研究奠定基础，我们将展示基因在培养犬细胞中的特异位点整合，并对基因组整合热点进行表征。这些实验将移动phiC31整合酶

DESCRIPTION (provided by applicant): A powerful new technology for gene therapy is now available. The technology uses an integrase enzyme from phage phiC31 that mediates chromosomal integration of therapeutic genes into a limited number of native sequences in mammalian genomes. This innovative technique is the only one that provides efficient site-specific, rather than random, chromosomal integration. The method also provides permanent, strong expression of the therapeutic gene. The integrase system has been validated in animal models in a number of tissues, species, and diseases. The focus of this proposal is to use the phiC31 integrase system to create a novel methodology for safe, simple, and permanent transfer of genes to the retina of larger mammals. We demonstrated an electroporation method that was effective for DNA delivery to rat retina and showed that integrase provided strong, long-term expression of a marker gene in the retinal pigment epithelium. Methods will now be established to achieve efficient gene delivery and site-specific integration into the retina of rabbits, as a model for later dog and human work. Innovative electroporation methods will be developed with collaborators from physics and ophthalmology to achieve surgical placement of electrodes to provide effective and precise delivery of therapeutic DNA to the RPE, with minimal tissue damage. The phiC31 integrase will be utilized to provide site-specific genomic integration and long-term expression of transferred genes in the retina. Delivery parameters will initially be optimized with in vitro model systems, while imaging of luciferase in the rabbit eye will be employed in long-term in vivo studies. To lay the groundwork for studies in the RPE65 dog to follow, site-specific integration into cultured dog cells will be demonstrated and genomic integration hotspots will be characterized. These experiments will move the phiC31 integrase