In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization.
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.
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.
Piotr J
PJ
Kamola
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Jeremy D A
JD
Kitson
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Gemma
G
Turner
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Klio
K
Maratou
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Sofie
S
Eriksson
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Aliza
A
Panjwani
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Linda C
LC
Warnock
GlaxoSmithKline R&D, Les Ulis 91951, France.
Gaelle A
GA
Douillard Guilloux
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Kitty
K
Moores
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Emma L
EL
Koppe
GlaxoSmithKline R&D, Upper Merion 19406, US.
William E
WE
Wixted
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Paul A
PA
Wilson
Department of Surgery and Cancer, Imperial College London, London SW7 2AZ, UK.
Nigel J
NJ
Gooderham
Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Harwell Science and Innovation Campus OX11 0RQ, UK.
Timothy W
TW
Gant
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Kenneth L
KL
Clark
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Stephen A
SA
Hughes
GlaxoSmithKline R&D, Stevenage SG1 2NY, UK.
Mark R
MR
Edbrooke
GlaxoSmithKline R&D, Ware SG12 0DP, UK.
Joel D
JD
Parry
D020137:Base Pair Mismatch; D002478:Cells, Cultured; D003198:Computer Simulation; D005091:Exons; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D006801:Humans; D007438:Introns; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000627:therapeutic use; D016914:Ribonuclease H / Q000378:metabolism
D016376:Oligonucleotides, Antisense; D016914:Ribonuclease H; C078856:ribonuclease HI