Abstract

Over the past decades the field of nucleic acid therapeutics has made substantial progress. Several recent regulatory approvals launched an expansion of the oligonucleotide drug (OND) field (1), exemplified by an increase in scientific publications and collaborations between academia and pharma. Antisense oligo (ASO) gapmers are 12–24 nucleotides long chemically modified single-stranded ONDs, which bind their cellular (m)RNA targets and subsequently recruit RNase H1 to degrade target RNA (2). Crucially, preclinical studies focusing on enhancing ASO gapmer functional delivery often neglect proper evaluation of ASO-induced toxicity. ASO-induced toxicities can be divided into three different categories: i) sequence dependent toxicity, ii) sequence and hybridization dependent toxicity, and iii) sequence and hybridization independent toxicity (3,4). It is hypothesized that the majority of ASOinduced toxicities is caused by sequence dependent ASO:protein interactions (5). Therefore, we aim to identify and molecularly characterize novel toxic ASO:protein interactions with the ultimate goal of developing a mechanistic sequence-specific ASO-induced toxicity model to aid rational ASO design. Novel toxic ASO:protein interactions will be identified through state-of-the-art biochemical, genetic and molecular/RNA biology techniques, including a whole genome CRISPR screen with mortality endpoint which will be performed in the second half of 2023. Here we present the essential preliminary validation steps for the whole genome CRISPR screen.References 1. Egli, M. and Manoharan, M. (2023) Nucleic Acids Research, 51, 2529-2573. 2. Crooke, S.T. et al. (2021) Nature Reviews Drug Discovery, 20, 427-453. 3. Andersson, P. (2022) Springer US, pp. 355-370. 4. Goyenvalle, A., et al. (2022) Nucleic Acid Ther, 31, 1-16. 5. Shen, W. et al. (2019) Nature Biotechnology, 37, 640-650.