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

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.

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.

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.

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.

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.