Could we delay photoreceptor loss in retinal degeneration disease by gene therapy?

在这项研究中，我们研究了病毒介导的基因转移是否可以延缓视网膜变性小鼠视网膜的光感受器丧失。我们使用的载体是重组腺相关病毒（rAAV），它的免疫原性较低，应该介导稳定的基因转染。我们在CMV启动子下纯化了rAAV靶基因，并注射到正常或视网膜变性模型小鼠的眼睛中。视网膜变性小鼠为cGMP磷酸二酯酶(PDE) γ亚基敲除小鼠，出生后21天内出现光感受器变性。我们通过光镜和电镜研究了rAAV注入眼内的情况，并分析了rAAV介导的基因在视网膜中的转移效率。报告基因（GFP）在正常小鼠视网膜中的表达通过玻璃体内注射在神经节细胞、内核层细胞和光感受器中表达，通过视网膜下注射在RPE细胞和光感受器中表达。注射后7天左右检测到GFP表达，并持续1个多月。在小鼠出生后5天左右，将含有cGMP PDEgamma亚基cDNA的0.5 μl 10^N感染单位/μl rAAV注入敲除小鼠的视网膜下间隙。注射1个月后，rAAV注射区存在3-5层光感受器，而对照组仅存在1层光感受器。电镜显示，存活的光感受器具有中等数量的外节，这在对照组中很少观察到。注射rAAV的视网膜未见炎症反应，也未见视网膜结构损伤。本研究表明，腺相关病毒介导的基因转移可延缓小鼠视网膜角膜变性光感受器的丧失。这意味着基因治疗色素性视网膜炎的可行性。对存活光感受器的功能研究、对rAAV的修饰以设置更多的存活细胞、无创地监测基因表达等方面的研究是必要的，我们正在为此而努力。少

In this study, we investigated if virus-mediated gene transfer could delay photoreceptor loss in retinal degeneration mouse retina. The vector we used is recombinant adeno-associated virus (rAAV), which is less immunogenic and is supposed to mediate stable gene transfection. We purified rAAV with target gene under CMV promoter, and injected into the eye of normal or retinal degeneration model mouse. The retinal degenerntion mouse is cGMP phosphodiesterase (PDE) gamma subunit knockout mouse, which shows photoreceptor degeneration within 21 days after birth. We studied rAAV injected eyes by light and electron microscopy and analyzed rAAV mediated gene transfer efficiency into the retina.1. reporter gene (GFP) expression in normal mouse retinaGFP is exprssed in ganglion cell, inner nuclear layer cell, and photoreceptors by intravitreal injection, and in RPE cells and photoreceptors by subretinal injection. GFP expression is detected around 7 days after injection and continued more than 1 … More momth.2. cGMP PDE gamma subunit gene expression in retinal degeneration mouse retinaWe injected 0.5 μl of 10^N infectious unit /μl rAAV with cGMP PDEgamma subunit cDNA into the subretinal space of knockout mouse around 5 days after birth. One month after injection, 3-5 layets of photoreceptors were observed to be present in rAAV injected area, compared with only 1 photoreceptor layer remained in control retina. Electron microscopy revealed that surviving photoreceptors possess moderate numbers of outer segments, which is little observed in control reina. Neither inflammation nor damage to the retinal structure was detected in rAAV injected retina.This study showed that the loss of photoreceptors in petinal degeneration mouse retina could be delayed by adeno-associated virus mediated gene transfer. This means the feasibility of gene therapy as the treatment for retinitis pigmentosa. More sudies, such as functional study of surviving photoreceptors, modification of rAAV to set more surviving cells and monitering the gene expression noninvasively, are necessary and we are now making efforts for them. Less