Chalberg Thomas W, Genise Hilary L, Vollrath Douglas, Calos Michele P
Department of Genetics, Stanford University School of Medicine, CA 94305, USA.
Invest Ophthalmol Vis Sci. 2005 Jun;46(6):2140-6. doi: 10.1167/iovs.04-1252.
Gene therapy has shown promise in animal models of retinal disease, with the most success achieved to date with viral vectors used for gene delivery. Viral vectors, however, have side effects and limitations and are difficult to manufacture. The present study was conducted in an attempt to develop a novel system for long-term gene transfer in rat retinal pigment epithelium (RPE), by using nonviral transfection methods for gene transfer and the integrase from the bacteriophage phiC31 to confer long-term gene expression by means of genomic integration.
Efficient nonviral delivery of plasmid DNA to rat RPE in vivo was achieved by using subretinal injection of plasmid DNA, followed by in situ electroporation. Gene delivery was evaluated by analyzing enhanced green fluorescent protein (eGFP) expression in frozen sections. In subsequent experiments, a plasmid expressing luciferase, with or without a plasmid encoding the phiC31 integrase, was delivered to rat RPE. Luciferase expression was followed over time by using in vivo luciferase imaging.
Subretinal injection followed by electroporation yielded abundant transgene expression in the rat RPE. Expression was strongest 48 hours after delivery. In the absence of phiC31 integrase, transgene expression declined to near-background levels within 3 to 4 weeks after treatment. By contrast, coinjection of the integrase plasmid led to long-term stable transgene expression throughout the 4.5-month test period. Eyes injected with phiC31 integrase showed approximately 85-fold higher long-term transgene expression in the retina than eyes without integrase.
Subretinal injection of DNA followed by electroporation affords abundant transfer of plasmid DNA in rat RPE. phiC31 integrase confers robust long-term transgene expression by mediating genomic integration of the transgene. These findings suggest that phiC31 integrase may be a simple and effective tool for nonviral long-term gene transfer in the eye.
基因治疗在视网膜疾病的动物模型中已显示出前景，迄今为止，用于基因递送的病毒载体取得了最大的成功。然而，病毒载体有副作用和局限性，且难以制造。本研究旨在开发一种用于大鼠视网膜色素上皮（RPE）长期基因转移的新系统，通过使用非病毒转染方法进行基因转移，并利用噬菌体phiC31的整合酶通过基因组整合实现长期基因表达。
通过视网膜下注射质粒DNA，随后进行原位电穿孔，实现质粒DNA在大鼠RPE体内的高效非病毒递送。通过分析冰冻切片中增强型绿色荧光蛋白（eGFP）的表达来评估基因递送。在后续实验中，将表达荧光素酶的质粒，无论有无编码phiC31整合酶的质粒，递送至大鼠RPE。通过体内荧光素酶成像随时间跟踪荧光素酶表达。
视网膜下注射后进行电穿孔在大鼠RPE中产生了丰富的转基因表达。递送后48小时表达最强。在没有phiC31整合酶的情况下，转基因表达在治疗后3至4周内降至接近背景水平。相比之下，共注射整合酶质粒导致在整个4.5个月的测试期内转基因长期稳定表达。注射phiC31整合酶的眼睛在视网膜中的长期转基因表达比未注射整合酶的眼睛高约8５倍。
视网膜下注射DNA后进行电穿孔可使质粒DNA在大鼠RPE中大量转移。phiC31整合酶通过介导转基因的基因组整合赋予强大的长期转基因表达。这些发现表明，phiC31整合酶可能是用于眼部非病毒长期基因转移的一种简单有效的工具。
