Zeng Yong, Takada Yuichiro, Kjellstrom Sten, Hiriyanna Kelaginamane, Tanikawa Atsuhiro, Wawrousek Eric, Smaoui Nizar, Caruso Rafael, Bush Ronald A, Sieving Paul A
National Institute on Deafness and Other Communication Disorders, Bethesda, MD 20892, USA.
Invest Ophthalmol Vis Sci. 2004 Sep;45(9):3279-85. doi: 10.1167/iovs.04-0576.
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.
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.
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.
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 postdevelopmental adult stage of XLRS disease.
创建并评估一种人类X连锁青少年视网膜劈裂症（XLRS）的小鼠模型，然后研究通过基因递送补充视网膜劈裂蛋白是否能逆转异常的“电阴性”视网膜电图（ERG）视网膜反应。
通过用新霉素抗性盒替代人类RS-1基因的小鼠同源物Rs1h的外显子1和1.6 kb内含子1，创建了一种X连锁视网膜劈裂小鼠（Rs1h-KO）模型。使用多克隆RS N端抗体通过免疫组织化学和蛋白质印迹分析评估RS蛋白。通过传统的全视野闪光ERG记录评估视网膜功能。通过用在巨细胞病毒（CMV）启动子调控下含有C57BL/6J Rs1h cDNA的腺相关病毒（AAV）（2/2）-CMV-Rs1h载体进行基因转移来评估RS蛋白补充疗法，并进行ERG功能分析。
在Rs1h-KO小鼠中，通过蛋白质印迹分析或免疫组织化学未检测到RS蛋白。暗适应ERG反应呈电阴性构型，Rs1h（- / Y）和Rs1h - / -小鼠的b波均降低，这是人类XLRS的典型特征。Rs1h-KO小鼠的组织学检查显示多个视网膜层结构紊乱，包括神经节细胞的重复和错位、通过内网状层的层间分离、外网状层的紊乱、外核层规则性丧失以及内/外节缩短和光感受器细胞核错位到该层。眼内注射AAV（2/2）-CMV-Rs1h后，免疫组织化学显示Rs1h（- / Y）小鼠的所有视网膜层均有视网膜劈裂蛋白表达，ERG记录显示电阴性波形逆转并恢复正常的正性b波。
RS-KO小鼠模拟了人类X连锁青少年视网膜劈裂症的结构特征，伴有多个视网膜层的分离和紊乱。Rs1h-KO功能缺陷导致电阴性ERG波形，这是人类视网膜劈裂症疾病的特征，并且表明在缺乏视网膜劈裂蛋白的情况下存在突触传递缺陷。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。这表明基因治疗即使在XLRS疾病的发育后成年阶段也是一种可行的治疗干预策略。
