Targeting RNA via small molecule exon skippers: development of high throughput RT-qPCR platform to assess targeted RNA.

Abstract: RNA plays a crucial role in various biological processes and diseases, making it a sensible target for several pharmaceutical companies. The most common way to target RNA is by employing antisense oligonucleotides (ASOs), an approach that preferentially targets unstructured regions. However, RNA activity is often mediated by the structure that it forms, making small molecules (SMs) a valuable alternative to ASOs, in addition to having a greater potential for oral bioavailability and blood-brain barrier penetrance.
Over the past decade new technical achievements layed the foundations for accelerating research on SMs targeting RNA, and the approval of the very first drug modulating splicing in 2020 (Risdiplam, sold under the brand name Evrysdi) paved the way into a renewed interest in this approach. The competitive pharmaceutical landscape indicates that targeting exon skipping is a promising approach to lowering a messenger RNA (mRNA) and protein of interest levels.
Reverse transcription quantitative PCR (RT-qPCR) is a powerful tool for the detection and quantification of RNA sequences. However, it is normally considered a low throughput method due to the cost of reagents and challenges associated with miniaturisation and automation, precluding high-throughput screenings. Herein we developed and validated a 384-well RT-qPCR workflow for a challenging target, a mRNA referred to here as target A. The goal of the project is to identify SMs that induce skipping of an exon (exon X) to generate a premature termination codon. This will lead to nonsense mediated decay of the transcript which will consequently reduce the level of mRNA and protein. This approach is precedented by exon skipping ASOs for target A.
By means of high dimensional experimentation (HDE), we systematically evaluated the formulation, concentration and volume of the critical reagents within the protocol, including in-house lysis conditions that ultimately reduced the cost by almost 60% compared to current standard practice. We utilized the latest technologies available in automated liquid handling to deliver a highly robust assay format suitable for design, make, test, analyse (DMTA) cycles in lead generation and optimisation stages, within 6 months from proof-of-concept initiation. Overall, the establishment of this platform enables a panel screening strategy to efficiently connect lead identification and lead optimization in parallel, with quantitative studies as part of routine drug discovery program support.