HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics

Abstract

Nucleic acid therapeutics are witnessing an impressive acceleration in recent years. They work through multiple mechanisms of action, including downregulation of gene expression and modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method to cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.

HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics

“…We tested the functionality of the PFlare5A-Tau10 mut reporter plasmid and of the HCS-Splice assay system using several splice modulators [51]. An example of one such modulator has been shown here to reduce exon 10 (E10+)-containing transcripts (Figure 4).…”