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。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。一些动物模型，自发突变体和基因工程动物已被确定为遗传性视网膜疾病。用于眼部疾病的绝大多数基因治疗方案涉及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，但病毒介导的基因治疗可能引起潜在的致命随机整合、免疫反应，并且在资金和时间上都具有高成本。非病毒基因治疗方法减少了许多这些不希望的影响，但表现出整合差，有限的转基因表达和降低的转导效率。最近，不可逆重组酶，噬菌体FC 31整合酶已被证明具有稳定的，位点特异性整合，可以促进稳定的，长期的转基因表达，具有携带大的DMA序列的能力。它可以通过非病毒技术，如电穿孔或脂质转染，这几乎没有副作用。该技术尚未应用于眼部疾病，特别是遗传性视网膜变性疾病。本文提出的研究旨在将该技术应用于由RPE 65基因突变引起的Leber先天性黑蒙小鼠模型，即Rpe 65-/-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良好的表征，并建立了功能测试参数。本提案的总体目标是利用噬菌体phiC 31整合酶技术来证明该动物模型中的功能恢复，从而为该疾病提供替代或补充疗法。进一步证明该技术可能应用于可能受益于基因治疗干预的其他遗传性眼病。

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.