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Abstract：
Leber congenital amaurosis (LCA) is a severe, genetically heterogeneous recessive eye disease in which ~ 35% of gene mutations are in-frame nonsense mutations coding for loss-of-function premature termination codons (PTCs) in mRNA. Nonsense suppression therapy allows read-through of PTCs leading to production of full-length protein. A limitation of nonsense suppression is that nonsense-mediated decay (NMD) degrades PTC-containing RNA transcripts. The purpose of this study was to determine whether inhibition of NMD could improve nonsense suppression efficacy in vivo. Using a high-throughput approach in the recessive cep290 zebrafish model of LCA (cep290;Q1223X), we first tested the NMD inhibitor Amlexanox in combination with the nonsense suppression drug Ataluren. We observed reduced retinal cell death and improved visual function. With these positive data, we next investigated whether this strategy was also applicable across species in two mammalian models: Rd12 (rpe65;R44X) and Rd3 (rd3;R107X) mouse models of LCA. In the Rd12 model, cell death was reduced, RPE65 protein was produced, and in vivo visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in Rd12 retina was through reduced UPF1 phosphorylation. In the Rd3 model, however, no beneficial effect was observed with Ataluren alone or in combination with Amlexanox. This variation in response establishes that some forms of nonsense mutation LCA can be targeted by RNA therapies, but that this needs to be verified for each genotype. The implementation of precision medicine by identifying better responders to specific drugs is essential for development of validated retinal therapies.
摘要：
Leber先天性黑蒙(LCA)是一种严重的,遗传异质性隐性眼病，其中约35％的基因突变是编码mRNA中功能丧失的过早终止密码子（PTC）的框内无义突变。无义抑制疗法允许PTC的通读,导致全长蛋白质的产生。无义抑制的限制是无义介导的衰变(NMD)降解含有PTC的RNA转录物。这项研究的目的是确定NMD的抑制是否可以改善体内无义抑制功效。在LCA的隐性ccep290斑马鱼模型中使用高通量方法（cep290;Q1223X），我们首先测试了NMD抑制剂Amlexanox与无义抑制药物Ataluren的组合。我们观察到视网膜细胞死亡减少和视觉功能改善。有了这些积极的数据，我们接下来研究了该策略是否也适用于两个哺乳动物模型的物种:Rd12(rpe65;R44X)和Rd3(rd3;R107X)LCA小鼠模型.在Rd12模型中，细胞死亡减少，产生RPE65蛋白，并改善了体内视觉功能测试。我们首次确定Amlexanox在Rd12视网膜中的作用机制是通过减少UPF1磷酸化。在Rd3模型中，然而,单独使用Ataluren或与Amlexanox组合未观察到有益效果。这种反应的变化表明，某些形式的无义突变LCA可以被RNA疗法靶向。但这需要验证每个基因型。通过识别对特定药物的更好应答者来实施精准医学对于开发经过验证的视网膜疗法至关重要。
