Wang Xia, Shan Xianghong, Gregory-Evans Kevin, Gregory-Evans Cheryl Y
Department of Ophthalmology and Visual Sciences, University of British Columbia, Vancouver BC V5Z 3N9, Canada.
Precis Clin Med. 2020 Jun;3(2):113-126. doi: 10.1093/pcmedi/pbaa009. Epub 2020 Mar 12.
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 . Using a high-throughput approach in the recessive zebrafish model of LCA (;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: (;R44X) and (;R107X) mouse models of LCA. In the model, cell death was reduced, RPE65 protein was produced, and visual function testing was improved. We establish for the first time that the mechanism of action of Amlexanox in retina was through reduced UPF1 phosphorylation. In the 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.
莱伯先天性黑蒙（LCA）是一种严重的、基因异质性隐性眼病，其中约35%的基因突变是框内无义突变，可编码mRNA中功能丧失的过早终止密码子（PTC）。无义抑制疗法可使PTC通读，从而产生全长蛋白质。无义抑制的一个局限性是无义介导的衰变（NMD）会降解含有PTC的RNA转录本。本研究的目的是确定抑制NMD是否能提高无义抑制效果。我们在隐性LCA斑马鱼模型（;Q1223X）中采用高通量方法，首先测试了NMD抑制剂氨来呫诺与无义抑制药物阿他芦醇联合使用的效果。我们观察到视网膜细胞死亡减少，视觉功能得到改善。基于这些阳性数据，我们接下来研究了该策略在两种哺乳动物模型中是否也适用于不同物种：LCA的（;R44X）和（;R107X）小鼠模型。在模型中，细胞死亡减少，产生了RPE65蛋白，视觉功能测试得到改善。我们首次确定氨来呫诺在视网膜中的作用机制是通过减少UPF1磷酸化。然而，在模型中，单独使用阿他芦醇或与氨来呫诺联合使用均未观察到有益效果。这种反应差异表明，某些形式的无义突变LCA可通过RNA疗法靶向治疗，但每种基因型都需要进行验证。通过识别对特定药物反应更好的患者来实施精准医学，对于开发经过验证的视网膜治疗方法至关重要。
