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Using RNA-seq to Assess Off-Target Effects of Antisense Oligonucleotides in Human Cell Lines

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Abstract

Background

The field of antisense oligonucleotide therapeutics is rapidly growing and in addition to small molecules and therapeutic antibodies, oligonucleotide-based gene expression modifiers have been developed as fully accepted therapeutics. Antisense oligonucleotides are designed to modify gene expression of their specific target genes. However, as their effect relies on Watson–Crick base pairing, they could also bind to other unintended complementary RNAs showing sufficient sequence homology, which in turn could lead to off-target effects. It is assumed that these off-target effects depend on the degree of complementarity between the antisense oligonucleotides and off-target sequences.

Objective

Aim of this study was the investigation of the effects of antisense oligonucleotides on the expression of potential off-targets having a defined number of mismatches to the oligonucleotide sequence.

Methods

We extend recent studies by investigating the off-target profile 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 significantly downregulated off-target genes for which one mismatch is present corroborates the requirement for intense bioinformatic screens and stringent specificity criteria to design antisense oligonucleotides with only minimal sequence complementarity to any non-target sequence.

Conclusions

Avoiding suppression of off-target genes by a thorough bioinformatics screen should strongly reduce the risk for toxicities caused by antisense oligonucleotide-mediated off-target RNA suppression and finally result in safer antisense oligonucleotide-based therapeutics.

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Acknowledgements

The authors thank Lisa Hinterwimmer, Monika Schell, and Stefanie Raith as well as the sequencing platform of the Centre National de Recherche en Génomique Humaine (CNRGH) for their excellent technical support.

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Authors

Corresponding authors

Correspondence to Sven Michel or Frank Jaschinski.

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Funding

No funding was received for the conduct of this study or the preparation of this article.

Conflict of interest

Sven Michel, Ksenia Schirduan, Richard Klar, and Frank Jaschinski are or were at the time the experiments were performed employees of Secarna Pharmaceuticals GmbH & Co. KG. Yimin Shen and Jörg Tost have no conflicts of interest that are directly relevant to the content of this article.

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Not applicable.

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Not applicable.

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Not applicable.

Code Availability

R code that was used do conduct the analysis is available on request from the corresponding author.

Data availability

The sequence data analyzed during the current study are available at the NCBI Sequence Read Archive under the BioProject accession number PRJNA668062.

Author contributions

SM planned the study and experiments, analyzed the data, performed the bioinformatic analysis, and wrote the manuscript. KS planned the study and experiments and performed the experiments. YS planned the RNA-seq experiments and performed the bioinformatic analysis. RK planned and performed the qPCR experiments and analysis. JT planned the RNA-seq experiments, interpreted the data, and wrote the manuscript. FJ planned the study, interpreted the data, and wrote the manuscript.

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Michel, S., Schirduan, K., Shen, Y. et al. Using RNA-seq to Assess Off-Target Effects of Antisense Oligonucleotides in Human Cell Lines. Mol Diagn Ther 25, 77–85 (2021). https://doi.org/10.1007/s40291-020-00504-4

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  • DOI: https://doi.org/10.1007/s40291-020-00504-4

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