Acute hepatotoxicity of 2′ fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins

Abstract

We reported previously that a 2′ fluoro-modified (2′ F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5–10–5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2′-O-(2-methoxyethyl) (2′ MOE) or 2′,4′-constrained 2′-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2′ F PS-ASO. Here, we report that 2′ F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2′ F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2′ F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2′ F nucleoside metabolites. Instead, we found that 2′ F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2′ F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2′ F PS-ASO in vivo.

Synthesis and evaluation of (S)-5′-C-aminopropyl and (S)-5′-C-aminopropyl-2′-arabinofluoro modified DNA oligomers for novel RNase H-dependent antisense oligonucleotides

“…For example, one kind of modication on phosphodiesters, phosphorothioate (PS), which is used in mipomersen and nusinersen, among others, could not only increase the nuclease resistance, but also enhance the intracellular internalization of ASOs, with the cytotoxicity of PS also determined in some of the cases. 2,3,[8][9][10] As in our previous reports, inserting an aminoalkyl side chain into the 4 0 -or 5 0 -site of nucleosides could signicantly increase the nuclease resistance of the resulting oligonucleotides. [11][12][13][14][15] Due to the decrease of nuclease resistance when the terminal amino moiety was protected by acetyl groups, it is assumed that the positive charge on the amino moiety of the aminoalkyl side chain acts as an obstacle to nucleases binding to oligonucleotides, and thus inhibits the degradation of oligonucleotides.…”

Interaction of modified oligonucleotides with nuclear proteins, formation of novel nuclear structures and sequence-independent effects on RNA processing

“…No caspase activation was observed 506 in the absence of intranuclear inclusions, identifying nuclear inclusion as complicit in 507 caspase activated apoptosis, leading to cell death. The tumour suppressor protein p53 was 508 decreased, following transfection with 2´ O-methyl phosphorothioate AOs, relative to 509 untreated control cells (Figure 5b and c), suggesting that the cell death observed was p53 510 independent, contrary to the findings by Shen et al, 2018(Shen et al 2018) when using a 2′ 511 fluoro-modified AO on a phosphorothioate backbone in the mouse. In comparison to 512 phosphorothioate AO transfection, p53 levels remained unchanged following PMO 513 transfection (Figure 5b and c).…”

“…Early work 85 indicated that negatively charged phosphorothioate AOs bind non-specifically to heparin-86 like proteins, laminin and collagen (Dias and Stein 2002) and more recently, 87 phosphorothioate AOs were reported to interact with nuclear paraspeckle-associated 88 proteins, sequestering them away from their endogenous target, the long non-coding RNA 89 NEAT1, and also seeding paraspeckle like-structures in the absence of NEAT1 (Shen et al 90 2014, Shen et al 2015). Paraspeckles and paraspeckle proteins are involved in 91 transcriptional regulation, transport and splicing pathways (Bond and Fox 2009, antisense oligonucleotides with a 5-10-5 gapmer configuration in mice (Shen et al 2018) 105…”

“…and while this study focused on the severity of 2´ fluoro-modified phosphorothioate AOs, all 106 2´ modifications evaluated have been shown to sequester paraspeckle proteins, albeit to 107 varying degrees (Shen et al 2014, Liang et al 2015, Shen et al 2018). The backbone-108 dependent binding of phosphorothioate-modified olionucleotides to platelets in vitro and in 109 vivo, mediated by the platelet-specific receptor glycoprotein VI (GPVI) is also of note (Flierl 110 et al 2015), considering the broad range of nucleic acid therapeutics currently under 111 investigation (for review see (Bennett et al 2017).…”

Strategies to target long non-coding RNAs in cancer treatment: progress and challenges

“…Sugar modifications to give a high affinity to nucleic acids cause increased off-target cleavage of ASOs and siRNAs [148][149][150]. This is because in this case, 1-2 mismatches are tolerable and hybridization can take place in shorter regions of homology [151]. Phosphorothioated oligonucleotides demonstrated pro-inflammatory properties [152].…”