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Novel EGFP reporter cell and mouse models for sensitive imaging and quantification of exon skipping

Authors: 
Hara Y, Mizobe Y, Inoue YU, Hashimoto Y, Motohashi N, Masaki Y, Seio K, Takeda S, Nagata T, Wood MJA, Inoue T, Aoki Y
Citation: 
Sci Rep. 2020;10(1):10110. doi: 10.1038/s41598-020-67077-4
Abstract: 
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.
Epub: 
Not Epub
Organism or Cell Type: 
cell culture, mice
Delivery Method: 
Endo-Porter, local injection