Journal Article

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

Takeda, Shin’ichi, Motohashi, Norio, Seio, Kohji, Aoki, Yoshitsugu, Hashimoto, Yasumasa, Hara, Yuko, Mizobe, Yoshitaka, Nagata, Tetsuya, Wood, Matthew J. A., Inoue, Takayoshi, Inoue, Yukiko U., Masaki, Yoshiaki

2020

Overview

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- mdx 23 Tg). Finally, we confirmed that the EGFP- mdx 23 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 13 / 14/35 / 631/154/152 / 631/61/32 / 64/60 / Animal models / Animals / Antisense oligonucleotides / Cell culture / Disease Models, Animal / Duchenne's muscular dystrophy / Dystrophin / Dystrophin - genetics / Dystrophy / Exon skipping / Exons - genetics / Exons - physiology / Female / Fluorescence / Frameshift mutation / Genes, Reporter - genetics / Genetic Therapy - methods / Green Fluorescent Proteins / Humanities and Social Sciences / Male / Mice / Mice, Inbred C57BL / Mice, Transgenic / Morpholinos - genetics / multidisciplinary / Muscle Fibers, Skeletal - metabolism / Muscular Dystrophy, Duchenne - genetics / Myotubes / Nucleic acids / Oligonucleotides - genetics / Oligonucleotides, Antisense - metabolism / Primary Cell Culture / RNA Splicing - genetics / RNA Splicing - physiology / Rodents / Science / Science (multidisciplinary) / Transgenic mice / Trinucleotide repeats