Retinal Gene Therapy by Site-Specific Integration

通过位点特异性整合进行视网膜基因治疗

• 批准号: 7122345
• 负责人: MICHELE P CALOS
• 金额: $ 15.44万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122345
• 关键词: animal genetic material tag; bacterial virus; biological models; biotechnology; electrodes; electroporation; eye surgery; gene expression; gene therapy; green fluorescent proteins; integrase; laboratory rabbit; luciferin monooxygenase; retina; retina disorder; technology /technique development; therapy design /development; tissue /cell culture; virus infection mechanism

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

描述(申请人提供)：一种强大的基因治疗新技术现已问世。这项技术使用了来自噬菌体phiC31的整合酶，它介导了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特定部位而不是随机的染色体整合的技术。该方法还提供了治疗性基因的永久、强表达。整合酶系统已经在许多组织、物种和疾病的动物模型中得到验证。这项建议的重点是使用phiC31整合酶系统来创建一种新的方法，用于将基因安全、简单和永久地转移到较大哺乳动物的视网膜。我们证明了一种有效的将DNA输送到大鼠视网膜的电穿孔方法，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而长期的表达。现在将建立有效的基因传递和定位整合到兔视网膜的方法，作为以后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的合作者一起开发，以实现电极的外科放置，以最小的组织损伤提供有效和准确的治疗性DNA输送到RPE。PhiC31整合酶将被用来提供特定部位的基因组整合和转移基因在视网膜中的长期表达。给药参数最初将通过体外模型系统进行优化，而荧光素酶在兔眼的成像将用于长期的体内研究。为了为接下来的RPE65犬的研究奠定基础，将展示特定部位整合到培养的狗细胞中，并将表征基因组整合的热点。这些实验将使phiC31整合酶