In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization
| Autor: | Jeremy D. A. Kitson, Kenneth L. Clark, Mark R. Edbrooke, Nigel J. Gooderham, Emma Koppe, William E. Wixted, Gaelle A. Douillard Guilloux, Linda C. Warnock, Joel D. Parry, Aliza Panjwani, Gemma Turner, Piotr J. Kamola, Paul A. Wilson, Klio Maratou, Stephen A. Hughes, Sofie Eriksson, Kitty Moores, Timothy W. Gant |
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| Rok vydání: | 2015 |
| Předmět: |
RNase P
Base Pair Mismatch In silico Ribonuclease H Computational biology Biology 03 medical and health sciences 0302 clinical medicine Chemical Biology and Nucleic Acid Chemistry Genetics Gene Knockdown Techniques Gene silencing Humans Computer Simulation Gene Silencing RNase H Cells Cultured 030304 developmental biology 0303 health sciences Messenger RNA Gene knockdown Oligonucleotide Exons Oligonucleotides Antisense Introns 030220 oncology & carcinogenesis biology.protein |
| Zdroj: | Nucleic Acids Research |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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