Vol.:(0123456789) Molecular Diagnosis & Therapy https://doi.org/10.1007/s40291-020-00504-4 ORIGINAL RESEARCH ARTICLE Using RNA‑seq to Assess Of‑Target Efects of Antisense Oligonucleotides in Human Cell Lines Sven Michel 1  · Ksenija Schirduan 1  · Yimin Shen 2  · Richard Klar 1  · Jörg Tost 2  · Frank Jaschinski 1 Accepted: 20 November 2020 © Springer Nature Switzerland AG 2020 Abstract Background The feld of antisense oligonucleotide therapeutics is rapidly growing and in addition to small molecules and therapeutic antibodies, oligonucleotide-based gene expression modifers have been developed as fully accepted therapeutics. Antisense oligonucleotides are designed to modify gene expression of their specifc target genes. However, as their efect relies on Watson–Crick base pairing, they could also bind to other unintended complementary RNAs showing sufcient sequence homology, which in turn could lead to of-target efects. It is assumed that these of-target efects depend on the degree of complementarity between the antisense oligonucleotides and of-target sequences. Objective Aim of this study was the investigation of the efects of antisense oligonucleotides on the expression of potential of-targets having a defned number of mismatches to the oligonucleotide sequence. Methods We extend recent studies by investigating the of-target profle of two 17-mer antisense oligonucleotides in two distinct human cell lines by a whole-transcriptome study using RNA sequencing. Results The relatively high percentage of signifcantly downregulated of-target genes for which one mismatch is present corroborates the requirement for intense bioinformatic screens and stringent specifcity criteria to design antisense oligonu- cleotides with only minimal sequence complementarity to any non-target sequence. Conclusions Avoiding suppression of of-target genes by a thorough bioinformatics screen should strongly reduce the risk for toxicities caused by antisense oligonucleotide-mediated of-target RNA suppression and fnally result in safer antisense oligonucleotide-based therapeutics. Supplementary information The online version of this article (https://doi.org/10.1007/s40291-020-00504-4) contains supplementary material, which is available to authorized users. * Sven Michel sven.michel@secarna.com * Frank Jaschinski frank.jaschinski@secarna.com 1 Secarna Pharmaceuticals, GmbH & Co. KG, Am Klopferspitz 19, 82152 Planegg/Martinsried, Germany 2 Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA-Institut de Biologie François Jacob, Evry, France Key Points RNA sequencing provides a useful tool to assess anti- sense oligonucleotide-mediated of-target efects. Applying RNA sequencing during the drug candidate selection process can result in the selection of safer candidates and can furthermore prevent candidates with a poor specifcity profle enter animal testing. The use of more than one cell line improves the outcome of RNA sequencing. 1 Introduction The feld of antisense oligonucleotide (ASO) therapeutics is rapidly growing and in addition to small molecules and therapeutic antibodies, oligonucleotide-based gene expres- sion modifers have developed as fully accepted therapeutics. By the end of 2019, seven treatments based on antisense oligonucleotides have been approved by the US Food and Drug Administration or the European Medicines Agency [1–7]. Although these are still based on lower afnity chem- istries, they pave the way for the latest generation of ASOs. There are diferent classes of ASOs that are all based on the Watson–Crick base pairing between the ASO and the tar- get sequence. One class targets splicing sites and for exam- ple aims to restore the generation of functional proteins. A