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.
Highlights
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene
antisense oligonucleotides (ASOs) candidates are screened by injecting them into the mouse skeletal muscles, collecting these tissue samples to evaluate the exon skipping by reverse transcription PCR (RT-PCR), and confirming the recovery of DMD expression by western blotting
We sought to develop a novel enhanced green fluorescence protein (EGFP)-mdx23/pCAGGS reporter vector to demonstrate the efficiency of specific ASOs for exon skipping
Summary
Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. ASO candidates are screened by injecting them into the mouse skeletal muscles, collecting these tissue samples to evaluate the exon skipping by reverse transcription PCR (RT-PCR), and confirming the recovery of DMD expression by western blotting. We demonstrated that we could screen highly potent chemical modifications of ASOs based on the intensities of EGFP signal using a plate reader With such an effective screening system, we revealed that we could precisely measure the exon skipping efficiency for candidate ASOs in a short time compared with the conventional method. We generated novel transgenic mice with the EGFP-based reporter (EGFP-mdx[23] Tg mice) to successfully detect the biodistribution of ASOs in vivo
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