non-viral medicated gene targeting to the retina

描述（由申请人提供）：遗传性视网膜疾病是失明的主要原因。已经鉴定出140多种不同致病基因的突变，其中60%以上已被克隆。一些动物模型，自发突变和基因工程动物已被确定为遗传性视网膜疾病。绝大多数眼部疾病的基因治疗方案涉及使用病毒介导的基因转移。病毒介导的基因治疗虽然具有高效的转导和强效的转基因表达，但可能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良影响，但表现出较差的整合，有限的转基因表达和降低转导效率。最近，一种不可逆重组酶噬菌体FC31整合酶被证明具有稳定的位点特异性整合，可以促进稳定、长期的转基因表达，并能够携带大的DMA序列。它可以通过非病毒技术来传递，比如电穿孔或脂肪感染，这些技术几乎没有副作用。这项技术尚未应用于眼部疾病，特别是遗传性视网膜退行性疾病。本文提出的研究旨在将该技术应用于由RPE65基因突变引起的Leber's Congenital Amaurosis小鼠模型，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，并建立了功能检测参数。本提案的总体目标是利用噬菌体phiC31整合酶技术在该动物模型中证明功能恢复，从而为该疾病提供替代或补充治疗。进一步证明该技术可能应用于其他遗传性眼病，这些疾病可能受益于基因治疗干预。

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.