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光感受器的丢失。我们使用的载体是重组腺相关病毒(RAAV)，它的免疫原性较低，被认为是介导稳定的基因转染。我们在CMV启动子的作用下，提纯了带有目的基因的重组腺病毒，并将其注射到正常或视网膜变性模型小鼠的眼内。视网膜退行性变小鼠是cGMP磷酸二酯酶(PDE)γ亚基基因敲除小鼠，出生后21天内表现为光感受器变性。我们用光镜和电子显微镜观察了rAAV注射眼的情况，分析了rAAV介导的视网膜基因转移效率。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，在RPE细胞和光感受器中通过视网膜下注射表达。注射后7天左右检测到绿色荧光蛋白的表达，并持续1…以上更多的月。CGMP PDEγ亚基基因在视网膜变性小鼠视网膜中的表达出生后5天左右，将携带cGMP PDEgamma亚基基因的0.5μL 10^N感染单位/μL重组腺病毒注射到基因敲除小鼠的视网膜下腔。注射后1个月，rAAV注射区有3~5层感光细胞，而对照视网膜仅有1层感光细胞。电子显微镜显示，存活的光感受器具有中等数量的外节，这在对照雷纳中很少观察到。注射rAAV的小鼠视网膜未见炎症反应，也未发现视网膜结构的损伤。本研究表明，腺相关病毒介导的基因转移可以延缓小鼠视网膜瓣膜退行性变中光感受器的丧失。这意味着基因治疗作为治疗视网膜色素变性的可行性。更多的研究，如存活光感受器的功能研究，修饰rAAV以建立更多的存活细胞，以及无创性地监测基因表达，都是必要的，我们正在努力。较少

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