Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping
Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping
复制标题
DOI：
10.1038/s41598-020-67077-4
复制
发表时间：
2020-06-22
影响因子：
4.6
通讯作者：
Aoki, Yoshitsugu
中科院分区：
文献类型：
--
作者：
Hara, Yuko;Mizobe, Yoshitaka;Aoki, Yoshitsugu
杜氏肌营养不良症（DMD）是由DMD基因无义或移码突变引起的一种致命的x连锁疾病。在多种治疗DMD的方法中，反义寡核苷酸（ASOs）介导的外显子跳变是一种很有前途的治疗方法。然而，对于成功的治疗，必须严格优化寡核苷酸化学以及ASOs的化学修饰。为了实现这一目标，我们的目标是开发一种新型的增强型绿色荧光蛋白（EGFP）报告分析系统，使我们能够对aso进行高效和高通量筛选。我们设计了一个新的CAG启动子表达载体，仅在ASOs诱导mdx型外显子23跳变时检测EGFP荧光。然后，使用磷酸二酯morpholino低聚物或不同LNA包络度的锁定核酸(LNA)/2 '-OMe混合器对C57BL/6原代肌管进行准确筛选。因此，我们利用该EGFP表达载体（EGFP-mdx23 Tg）构建了一种新的转基因小鼠模型。最后，我们证实EGFP-mdx23 Tg为检查ASOs的有效性和生物分布提供了一个高度敏感的平台，用于外显子跳过治疗。因此，该分析系统提供了一个简单而高度敏感的平台来优化寡核苷酸化学以及ASOs的化学修饰。
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2 '-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs.