应用类器官芯片研究Laminin在USH2A突变型视网膜色素变性发病中的作用及机制

Retinitis pigmentosa (RP) is a type of progressive blinding eye disease inherited by a single gene. In the Asian population, USH2A is a common mutant gene that causes RP, but its pathogenesis is unknown and treatment options are limited. Our previous experimental studies showed that USH2A mutant retinal organoids have abnormal morphological characteristics, which include delayed self-assembly of organoids, delayed differentiation of neuroepithelial layers, and significant down-regulation of laminin expression. However, the morphology of retinal organoids can be significantly improved through intervention to increase laminin expression. These results suggest that USH2A mutation may affect the development of retinal organoids by acting on the expression of Laminin. Therefore, this project establishes an individualized cellular disease model of patients with USH2A mutant RP through retinal organoids. The perfusion organoid chip system was used to optimize the differentiation of USH2A mutant retinal organoids and determine their regularity. Combined with transcriptome sequencing and differential molecular target interventions, the morphological, molecular and functional abnormalities of retinal organoids are dynamically detected. The role and molecular mechanism of Laminin in the development of retinal organoid with USH2A mutant is analyzed. At the same time, individualized drugs for RP disease of USH2A are screened to explore therapeutic targets. The overall impacts of this project include elucidating the molecular mechanism of RP onset and progression, exploring the correlation among abnormal protein expression resulting from RP mutations, RP clinical characteristics, and the phenotype of retinal organoid derived from RP patients. This will contribute greatly to our knowledge of RP disease development with USH2A mutant and offer novel insights on new treatment strategies.

视网膜色素变性(retinitis pigmentosa,RP)是一类单基因遗传的致盲性眼病。在我国人群中，USH2A是导致RP的常见突变基因，但其发病机制未明，治疗手段有限。我们前期研究显示：USH2A突变型视网膜类器官的形态异常与下调laminin表达量相关；而上调laminin的表达可以改善其病变类器官的形态结构。因此，本项目通过USH2A突变型RP患者的视网膜类器官疾病模型，利用灌流类器官芯片优化USH2A突变型视网膜类器官的分化并阐明其规律；结合转录组测序和差异分子靶点干预，动态分析视网膜类器官细胞的形态、分子及功能异常特征，阐明laminin在USH2A突变型视网膜类器官发育过程中的作用及分子机制，筛选相应的个体化药物，探索治疗靶点。明确Laminin在USH2A型基因突变-视网膜类器官疾病模型-患者临床表征的作用规律。为临床早期诊断、干预及治疗USH2A型RP提供新的思路。

遗传性视网膜变性疾病 (hereditary retinal diseases, HRD) 是一种遗传的视网膜退行性疾病，在全球范围内，HRD是导致严重视觉功能障碍的主要原因，其发病率约为1/3000。在我国，USH2A (c.8559-2A>G) 位点突变是RP和II型Usher综合征 (Usher syndrome type II, USH II)型视网膜变性疾病的高频突变位点。这两种疾病都属于常染色体隐性遗传，相同临床表征为夜盲，视野缺损和进行性视功能障碍，最终失明。视功能障碍不但对患者的日常生活和工作造成很大影响，也会加重家庭和社会的负担，由此带来严重的社会和经济问题。但是，目前对于USH2A(c.8559-2A>G)突变位点在RP/USH II视网膜变性疾病的致病机制和发病规律仍不清楚，缺乏有效的治疗手段。本项目通过招募USH2A型RP/USH II患者，利用体细胞重编程技术在体外建立USH2A (c.8559-2A>G)突变型RP/USH II患者诱导多能干细胞(induced pluripotent stem cell, iPSC)的细胞系，明确USH2A (c.8559-2A>G)型RP/USH II患者的临床表征，研究USH2A (c.8559-2A>G)突变对病变iPSCs的影响；而后进一步分化成为视网膜类器官(ROs)疾病模型，搭建ROs芯片共培养体系，延长患者来源ROs的培养时间，研究USH2A (c.8559-2A>G)突变型RP/USH II疾病的发生机制。此外，我们通过构建了敲入型Ush2ac.8559-2A>G小鼠模型，验证明确USH2A (c.8559-2A>G)突变导致RP/USH II型疾病发生发展的分子机制。本研究为研究USH2A (c.8559-2A>G)突变型RP/USH II的临床表征提供理论依据，也为临床精准干预及治疗视网膜变性疾病提供新的思路。

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