武汉云克隆科技股份有限公司

Abstract: X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

摘要：X连锁的青少年视网膜劈裂症（XLRS）是由RS1基因突变引起的视网膜退行性病症，其编码称为视黄酸软骨素的蛋白质。该疾病是基因替代疗法的优秀候选者，因为大多数突变已显示导致视网膜结构中分泌蛋白的完全缺乏。在这项工作中，我们研究了基于固体脂质纳米粒（SLN）的非病毒载体在玻璃体内给予Rs1h缺陷小鼠后在光感受器（PR）中诱导视网膜色素的表达的能力。我们设计了两种用SLN，鱼精蛋白和葡聚糖（DX）或透明质酸（HA）制备的载体，载有含有在鼠视蛋白启动子（mOPS）控制下的人RS1基因的质粒。在体外，纳米载体能够诱导PR细胞系中视网膜色素的表达。在注射到鼠玻璃体后，用HA制备的制剂在PR中诱导比用DX制备的制剂更高的PR转染水平。此外，双极细胞所在的内核层（INL）中的视黄素水平也较高。玻璃体注射给予Rs1h缺陷小鼠两周后，两种制剂通过诱导空洞和PR损失的减少以及视网膜和外核层（ONL）厚度的增加显示视网膜结构的显着改善。 HA-SLN导致视网膜和ONL厚度的显着增加，这可以通过PR的更高转染水平来解释。总之，我们已经显示了在通过基于SLN的非病毒载体成功递送后，Rs1h缺陷小鼠的视网膜的结构改善，其中PR特异性表达由特异性启动子mOPS驱动的RS1基因。

使用试剂原文信息：Enzyme-linked Immunosorbent Assay (ELISA) Kit for the detection of retinoschisin was obtained from USCN Life Science Inc. (Houston, USA).