Covello, G.; Siva, K.; Adami, V.; Denti, M.A. HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics. Cells2023, 12, 1959.
Covello, G.; Siva, K.; Adami, V.; Denti, M.A. HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics. Cells 2023, 12, 1959.
Covello, G.; Siva, K.; Adami, V.; Denti, M.A. HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics. Cells2023, 12, 1959.
Covello, G.; Siva, K.; Adami, V.; Denti, M.A. HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics. Cells 2023, 12, 1959.
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.
Keywords
Alternative Splicing; two-colour (GFP/RFP) Fluorescent Reporter; MAPT; Exon-Skipping; FTDP-17; High Content Screening; siRNA; Nucleic Acids Therapeutics; Drug discovery
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
