Table 1.
Hybridization dependent off-target effects of antisense oligonucleotides
| mRNA target | Off-target genes/effects | Comments: Cell culture/animal model/phase of clinical study | Nucleic acid modifications | Intracellular or tumor delivery/concentration | Assessment method | Ref. |
|---|---|---|---|---|---|---|
| Human apolipoprotein C3 (ApoC3) |
Genes associated with hepatotoxicity as well as 17 genes involved in the clathrin-mediated endocytosis. | mice | LNA-DNA gapmer |
25 mg/kg (5.7µM) |
Microarray | 41 |
| BACH1 | mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. | A549 and NHBE cells | LNAa-PSb ASOc gapmerd |
unassisted or Lipofectamine 2000, Up to 50 µM | qPCR, branched DNA assay, immunoblotting | 42 |
| F7, F11 and SOD1 | hepatotoxicity mediated by silencing of many unintended transcripts through RNase H1 cleavage. | mice |
LNA-PS ASO gapmers, cEt-PS ASO gapmers | Subcutaneous injection | qPCR, microarray analysis, western blot | 43 |
| Nr3c, Acsl1, ApoB, Hprt1, and human Kif11 | hepatotoxicity not linked with on-target activity of ASO agents, but with the RNase H1 cleavage activity. | mice | LNA-PS ASO gapmers | Subcutaneous injection | qPCR, microarray analysis, western blot | 44 |
| ApoB, Pcsk9 | Significant and specific reduction of many transcripts with one- or two mismatches or bulges by RNase H1 mediated cleavage activity. | mice | LNA-PS ASO gapmers | Intravenous injection | qPCR, microarray analysis | 45 |
| Xbra, Xbra3 | Splicing defects in dtymk and abi1 genes contained ≥8 base pairs complementarity to PMO. | Xenopus tropicalis | PMOe | qPCR | 20 | |
| Myd88 | Hepatotoxic ASO-LNA demonstrated downregulation of 293 off-target genes and upregulation of 60 off-target genes and concentration dependent elevation of caspase activity in 3T3 cell line, induction of caspase activity in A549, HeLa, HepG2 cells. | 3T3, A549, HeLa, HepG2 cells | LNA | Lipofectamine 2000, 3-100 nM | Caspase 3/7 activity measurements, qPCR, microarray analysis | 47 |
aLNA - locked nucleic acid modification;
bPS - phosphorothioate modification;
cASO - antisense oligonucleotide;
dgapmer - ASO consisting of internal DNA gap flanked with LNA modified nucleotides;
ePMO - phosphorodiamidate morpholino modified ASO.