RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis
RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis
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DOI：
10.1167/iovs.04-0576
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发表时间：
2004-09-01
影响因子：
4.4
通讯作者：
Sieving, PA
中科院分区：
文献类型：
--
作者：
Zeng, Y;Takada, Y;Sieving, PA
目的。目的建立和评价人X连锁幼年视网膜劈裂症(XLRS)小鼠模型，探讨基因治疗能否逆转视网膜电信号(ERG)异常反应。方法：用新霉素抗性盒替代人RS-1基因的外显子1和1.6kb的内含子1，建立X连锁视网膜劈裂小鼠(Rs1h-KO)模型。用多克隆RS N末端抗体进行免疫组织化学和Western印迹分析。视网膜功能通过常规的全视野闪光ERG记录进行评估。通过携带C57BL/6J Rs1h基因的AAV(2/2)-CMV-Rs1h载体在CMV启动子的调控下进行RS蛋白治疗，并进行ERG功能分析。结果：免疫印迹分析和免疫组织化学检测未检测到RS蛋白。暗适应的ERG反应呈电负性，Rs1h(-/Y)和Rs1h(-/-)小鼠的b波减少，这是人类XLRS的典型特征。组织学检查显示Rs1h-KO小鼠视网膜多层结构紊乱，包括神经节细胞的复制和错位，内网状层的板层剥离，外网状层的解体，外核层失去规律性，内外节段缩短，感光核错定位于该层。眼内注射AAV(2/2)-CMV-Rs1h后，免疫组织化学显示Rs1h(-/Y)小鼠视网膜各层都有视黄醇的表达，ERG记录显示电负性波形逆转，恢复正常的正b波。结论RS-KO小鼠模拟人X连锁幼年视网膜劈裂症的结构特征，通过多层视网膜剥离和剥离。Rs1h-KO功能缺陷导致电阴性ERG波形，这是人类视网膜劈裂疾病的特征，并暗示在缺乏视黄蛋白的情况下存在突触传递障碍。在成年Rs1h-KO小鼠中，补充正常Rs1h蛋白的替代疗法恢复了正常的ERG构型。这表明，即使在XLRS病的发育后成人阶段，基因治疗也是一种可行的治疗干预策略。
PURPOSE. To create and evaluate a mouse model of human X-linked juvenile retinoschisis (XLRS) and then investigate whether supplementing with the retinoschisin protein by gene delivery can reverse the abnormal "electronegative" electroretinogram ( ERG) retinal response.METHODS. An X-linked retinoschisis mouse (Rs1h-KO) model was created by substituting a neomycin resistance cassette for exon 1 and 1.6 kb of intron 1 of Rs1h, the murine orthologue of the human RS-1 gene. RS protein was evaluated by immunohistochemistry and Western blot analysis with a polyclonal RS N-terminus antibody. Retinal function was evaluated by conventional, full-field flash ERG recordings. RS protein supplementation therapy was evaluated by gene transfer with an AAV(2/2)-CMV-Rs1h vector containing C57BL/6J Rs1h cDNA under the regulation of a CMV promoter, and ERG functional analysis was performed.RESULTS. No RS protein was detected by Western blot analysis or immunohistochemistry in the Rs1h-KO mouse. Dark-adapted ERG responses showed an electronegative configuration, with b-wave reduction in both Rs1h(-/Y) and Rs1h(-/-) mice, typical of XLRS in humans. Histologic examination of Rs1h-KO mice showed disorganization of multiple retinal layers, including duplication and mislocalization of ganglion cells, laminar dissection through the inner plexiform layer, disorganization of the outer plexiform layer, loss of regularity of the outer nuclear layer, and shortening of the inner/outer segments with mislocalization of photoreceptor nuclei into this layer. After intraocular administration of AAV(2/2)-CMV-Rs1h, immunohistochemistry showed retinoschisin expression in all retinal layers of Rs1h(-/Y) mice, and ERG recordings showed reversal of the electronegative waveform and restoration of the normal positive b- wave.CONCLUSIONS. The RS-KO mouse mimics structural features of human X-linked juvenile retinoschisis with dissection through, and disorganization of, multiple retinal layers. The Rs1h-KO functional deficit results in an electronegative ERG waveform that is characteristic of human retinoschisis disease and that implicates a synaptic transmission deficit in the absence of retinoschisin protein. Replacement therapy by supplementing normal Rs1h protein in the adult Rs1h-KO mouse restored the normal ERG configuration. This indicates that gene therapy is a viable strategy of therapeutic intervention even in the post-developmental adult stage of XLRS disease.