Abstract
Splice-switching oligonucleotides (SSOs) that can modulate RNA splicing are used for the treatment of many genetic disorders. To enhance the efficacy of modulating splicing, it is important to optimize SSOs with regard to target sites, GC content, melting temperature (Tm value), chemistries, and lengths. Thus, in vitro assay systems that allow for the rapid and simple screening of SSOs are essential for optimizing SSO design. In this study, we established a novel tri-chromatic reporter cell line for SSO screening. This reporter cell line is designed to express three different fluorescent proteins (blue, green, and red) and was employed for high content screening (HCS, also known as high content analysis; HCA) for the evaluation of SSO-induced exon skipping by analyzing the expression levels of fluorescent proteins. The blue fluorescent protein is stably expressed throughout the cell and is useful for data normalization using cell numbers. Furthermore, both the green and red fluorescent proteins were used for monitoring the splicing patterns of target genes. Indeed, we demonstrated that this novel reporter cell line involving HCS leads to a more rapid and simple approach for the evaluation of exon skipping than widely used methods, such as RT-PCR, western blotting, and quantitative RT-PCR. Additionally, a brief screening of Locked nucleic acids (LNA)-based SSOs targeting exon 51 in DMD was performed using the reporter cell line. The LNA-based SSO cocktail shows high exon 51 skipping in a dose-dependent manner. Furthermore, the LNA-based SSO cocktails display high exon 51 skipping activities on endogenous DMD mRNA in human rhabdomyosarcoma cells.
Highlights
Antisense-mediated splicing modulation is an attractive therapeutic approach for many genetic disorders involving RNA mis-splicing [1]
This study aimed to develop a novel tri-chromatic reporter cell line for the evaluation of the exon skipping activity of Locked nucleic acids (LNA)-based splice-switching oligonucleotides (SSOs) that could be applied for HCS
We performed a brief screening of LNA-based SSOs targeting Duchenne muscular dystrophy (DMD) exon 51 and revealed that a cocktail of LNA-based SSOs shows a higher exon skipping activity
Summary
Antisense-mediated splicing modulation is an attractive therapeutic approach for many genetic disorders involving RNA mis-splicing [1]. A previous study revealed that over 60% of point mutations result in splicing errors [2]. In 2016, the US Food and Drug Administration (FDA) approved two splice-switching oligonucleotides (SSOs): eteplirsen for the Duchenne muscular dystrophy (DMD) and nusinersen for spinal muscular atrophy (SMA). Reporter cell line for the rapid screening of splice-switching oligonucleotides using HCS had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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