Alport syndrome, a disease of kidney, ear, and eye, is caused by pathogenic variants in the COL4A3, COL4A4, or COL4A5 genes encoding collagen α3α4α5(IV) of basement membranes. Collagen IV chains that are truncated due to nonsense variants/premature termination codons (PTCs) cannot assemble into heterotrimers or incorporate into basement membranes. To investigate the feasibility of PTC readthrough therapy for Alport syndrome, we utilized two NanoLuc reporters in transfected cells: full-length for monitoring translation, and a split version for assessing readthrough product function. Full-length assays of 49 COL4A5 nonsense variants identified eleven as susceptible to PTC readthrough using various readthrough drugs. In split-NanoLuc assays, the predicted missense α5(IV) readthrough products of five nonsense mutations could heterotrimerize with α3(IV) and α4(IV). Readthrough was also observed in kidney cells from an engineered Col4a5 PTC mouse model. These results suggest that readthrough therapy is a feasible approach for a fraction of patients with Alport syndrome.

Alport 综合征是一种肾脏、耳朵和眼睛疾病，由编码基底膜胶原蛋白 α3α4α5(IV) 的COL4A3、COL4A4或COL4A5基因的致病变异引起。由于无义变体/过早终止密码子 (PTC) 而被截断的胶原蛋白 IV 链不能组装成异源三聚体或并入基底膜。为了研究 PTC 通读治疗 Alport 综合征的可行性，我们在转染细胞中使用了两个 NanoLuc 报告基因：全长用于监测翻译，以及用于评估通读产品功能的拆分版本。49 个COL4A5的全长检测无意义变体使用各种通读药物确定了 11 个对 PTC 通读敏感。在 split-NanoLuc 分析中，五种无义突变的预测错义 α5(IV) 通读产物可以与 α3(IV) 和 α4(IV) 异三聚化。在工程化Col4a5 PTC 小鼠模型的肾细胞中也观察到了通读。这些结果表明，对于部分 Alport 综合征患者，通读疗法是一种可行的方法。