non-viral medicated gene targeting to the retina

描述(申请人提供)：遗传性视网膜疾病是导致失明的主要原因。目前已鉴定出140多个不同致病基因的突变，其中60%以上已被克隆。几种动物模型，包括自发突变动物和基因工程动物，已经被确定为遗传性视网膜疾病。绝大多数眼科疾病的基因治疗方案都涉及到使用病毒介导的基因转移。虽然病毒介导的基因治疗在转导和有效的转基因表达方面是有效的，但它可以引起潜在的致命的随机整合和免疫反应，并且在资金和时间上都有很高的成本。非病毒基因治疗方法减少了许多这些不必要的影响，但表现出整合能力差、转基因表达受限和转导效率降低的特点。最近，一种不可逆的重组酶，噬菌体FC31整合酶被证明具有稳定的、位点特异性的整合，可以促进稳定的、长期的转基因表达，并能够携带大的DMA序列。它可以通过电穿孔或脂质体等非病毒技术传递，这些技术几乎没有副作用。这项技术还没有应用于眼部疾病，特别是遗传性视网膜退行性疾病。这里提出的研究旨在将这项技术应用于Leber先天性巨结肠的小鼠模型，该模型由RPE65基因突变引起，即RPE65-/-小鼠。病毒介导的基因治疗在该模型中实现了功能恢复，随后对该模型进行了很好的表征，并建立了功能测试参数。这项建议的总体目标是利用噬菌体phiC31整合酶技术来证明这种动物模型的功能恢复，从而为这种疾病提供一种替代或补充治疗方法。进一步证明这项技术可能应用于其他可能受益于基因治疗干预的遗传性眼病。

DESCRIPTION (provided by applicant): Inherited retinal diseases are a major cause of blindness. Mutations in over 140 different causative genes have been identified, of which over 60% have been cloned. Several animal models, both spontaneous mutants and genetically engineered animals have been identified for inherited retinal diseases. The vast majority of gene therapy protocols for ocular disease involve the use of viral mediated gene transfer. Although efficient in transduction and potent transgene expression, viral mediated gene therapy can cause potentially lethal random integration, immunological reaction, and has a high cost in both funding and time. Non-viral gene therapy approaches reduce many of these unwanted effects, but exhibits poor integration, limited transgene expression and decreased transduction efficiency. Recently, an irreversible recombinase, phage FC31 integrase has been shown to have stable, site-specific integration that can promote stable, long-term transgene expression, with the ability to carry large DMA sequences. It can be delivered through non-viral techniques, such as electroporation or lipofection, which have few side effects. This technology has not been applied to ocular disease, and in particular hereditary retinal degenerative disease. The research proposed here aims to apply this technology to a murine model for Leber's Congenital Amaurosis, caused by mutations in the RPE65 gene, the Rpe65-/- mouse. Viral mediated gene therapy has achieved function recovery in this model, subsequently the model has been well characterized, and functional testing parameters have been established. The overall aim of this proposal to utilize phage phiC31 integrase technology to demonstrate functional recovery in this animal model, and thereby offer an alternative or complementary therapy for this disease. Further to demonstrate possible application of this technology to other inherited ocular diseases that may benefit from gene therapy intervention.