In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization

Piotr J. Kamola*, Jeremy D.A. Kitson, Gemma Turner, Klio Maratou, Sofie Eriksson, Aliza Panjwani, Linda C. Warnock, Gaelle A. Douillard Guilloux, Kitty Moores, Emma L. Koppe, William E. Wixted, Paul A. Wilson, Nigel J. Gooderham, Timothy Gant, Kenneth L. Clark, Stephen A. Hughes, Mark R. Edbrooke, Joel D. Parry

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

71 Citations (Scopus)


With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries'may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.

Original languageEnglish
Pages (from-to)8638-8650
Number of pages13
JournalNucleic Acids Research
Issue number18
Publication statusPublished - 15 Oct 2015

Bibliographical note

Funding Information:
EPSRC Industrial CASE Award (Imperial College London, GlaxoSmithKline and Public Health England) (EP/J502017/1 to P.J. Kamola). Funding for open access charge: GlaxoSmithKline.

Publisher Copyright:
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


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