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

Autor:	Kamola PJ; Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, UK GlaxoSmithKline R&D, Ware SG12 0DP, UK GlaxoSmithKline R&D, Stevenage SG1 2NY, UK Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Harwell Science and Innovation Campus OX11 0RQ, UK piotr.j.kamola@gsk.com., Kitson JD; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Turner G; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Maratou K; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Eriksson S; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Panjwani A; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Warnock LC; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Douillard Guilloux GA; GlaxoSmithKline R&D, Les Ulis 91951, France., Moores K; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Koppe EL; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Wixted WE; GlaxoSmithKline R&D, Upper Merion 19406, US., Wilson PA; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Gooderham NJ; Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, UK., Gant TW; Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Harwell Science and Innovation Campus OX11 0RQ, UK., Clark KL; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Hughes SA; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Edbrooke MR; GlaxoSmithKline R&D, Stevenage SG1 2NY, UK., Parry JD; GlaxoSmithKline R&D, Ware SG12 0DP, UK.
Jazyk:	angličtina
Zdroj:	Nucleic acids research [Nucleic Acids Res] 2015 Oct 15; Vol. 43 (18), pp. 8638-50. Date of Electronic Publication: 2015 Sep 03.
DOI:	10.1093/nar/gkv857
Abstrakt:	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. (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
Databáze:	MEDLINE
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