In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA‐splicing competent minigenes, we also show that the ExspeU1‐mediated splicing correction at the RNA level recovered the full‐length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1‐mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease‐causing CFTR mutations.

中文翻译：

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用修饰的 U1 snRNA 拯救囊性纤维化中常见的外显子跳跃突变。

在囊性纤维化 (CF) 中，剪接缺陷的纠正代表了一种有趣的治疗方法，可以恢复正常的CFTR功能。在本研究中，我们重点研究了 10 个常见突变/变体：711+3A>G/C、711+5G>A、TG13T3、TG13T5、TG12T5、1863C>T、1898+3A>G、2789+5G>A 和 3120G >A 导致跳过相应的CFTR外显子 5、10、13、16 和 18。为了挽救剪接缺陷，我们在小基因检测中测试了一组修饰的 U1 小核 RNA (snRNA)，命名为外显子特异性 U1 (ExSpeU1s)，经过工程改造以结合每个缺陷外显子下游的内含子序列。使用这种方法，我们表明分析的所有 10 个剪接突变都被特定的 ExSpeU1s 有效纠正。使用互补的 DNA 剪接能力小基因，我们还表明，在 RNA 水平上 ExspeU1 介导的剪接校正恢复了全长CFTR1863C>T、1898+3A>G、2789+5G>A 变体的蛋白质。此外，在外显子 13 上进行的详细诱变实验使我们确定了一个新的内含子调控元件，该元件参与了 ExSpeU1 介导的剪接拯救。这些结果提供了一种基于修饰的 U1 snRNA 的通用策略，以纠正一组致病CFTR突变中的外显子跳跃。