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Impact of Oligonucleotide Structure, Chemistry, and Delivery Method on In Vitro Cytotoxicity
Maja M Janas1, Yongfeng Jiang1, Mark K Schlegel1
1Alnylam Pharmaceuticals, Inc. , Cambridge, Massachusetts.
Abstract:
Single-stranded (ss) 2'-fluoro (2'-F)-modified oligonucleotides (ONs) with a full phosphorothioate (PS) backbone have been reported to be cytotoxic and cause DNA double-strand breaks (DSBs) when transfected into HeLa cells. However, the molecular determinants of these effects have not been fully explored. In this study, we investigated the impact of ON structure, chemistry, delivery method, and cell type on in vitro cytotoxicity and DSBs. We found that ss PS-ONs were more cytotoxic than double-stranded (ds) PS-ONs, irrespective of the 2'-ribose chemistry, inclusive of the 2'-F modification. Cytotoxicity of ss ONs was most affected by the total PS content, with an additional contribution of 2'-F substitutions in HeLa, but not HepG2, cells. The relatively mild cytotoxicity of ds ONs was most impacted by long contiguous PS stretches combined with 2'-F substitutions. None of the tested ds 2'-F-modified PS-ONs caused DSBs, while the previously reported DSBs caused by ss 2'-F-modified PS-ONs were PS dependent. HeLa cells were more sensitive to ON-mediated toxicity when transfected with Lipofectamine 2000 versus Lipofectamine RNAiMax. Importantly, asialoglycoprotein receptor-mediated uptake of N-acetylgalactosamine-conjugated ss or ds PS-ONs, even those with long PS stretches and high 2'-F content, was neither cytotoxic nor caused DSBs at transfection-equivalent exposures. These results suggest that in vitro cytotoxicity and DSBs associated with ONs are delivery method dependent and primarily determined by single-stranded nature and PS content of ONs.
Insights
Single-stranded oligonucleotides (ONs) with phosphorothioate (PS) backbones are cytotoxic and cause DNA double-strand breaks (DSBs), but delivery method significantly impacts these effects.
Area of Science:
- Oligonucleotide chemistry and delivery
- Molecular toxicology
- Cellular uptake mechanisms
Background:
- Single-stranded (ss) 2'-fluoro (2'-F)-modified oligonucleotides (ONs) with phosphorothioate (PS) backbones can be cytotoxic and induce DNA double-strand breaks (DSBs).
- The specific molecular factors driving these cytotoxic effects remain incompletely understood.
Purpose of the Study:
- To investigate how oligonucleotide (ON) structure, chemistry, delivery method, and cell type influence in vitro cytotoxicity and DSBs.
- To elucidate the determinants of ON-induced toxicity and DNA damage.
Main Methods:
- Comparison of cytotoxicity and DSB induction between single-stranded (ss) and double-stranded (ds) PS-ONs with varying 2'-F modifications.
- Assessment of different transfection reagents (Lipofectamine 2000 vs. RNAiMax) and cell types (HeLa vs. HepG2).
- Evaluation of N-acetylgalactosamine (GalNAc)-conjugated ONs via asialoglycoprotein receptor (ASGPR)-mediated uptake.
Main Results:
- ss PS-ONs were more cytotoxic than ds PS-ONs, with cytotoxicity influenced by PS content and 2'-F substitutions (cell-type dependent).
- ds ON cytotoxicity was affected by long PS stretches and 2'-F modifications; ds ONs did not cause DSBs.
- ASGPR-mediated uptake of GalNAc-conjugated ONs showed no cytotoxicity or DSBs, irrespective of ss/ds nature, PS content, or 2'-F modification.
Conclusions:
- In vitro cytotoxicity and DSBs from ONs are primarily dependent on their single-stranded nature and phosphorothioate (PS) backbone content.
- The delivery method is a critical determinant of ON-induced toxicity and DNA damage.
- Targeted delivery via ASGPR-mediated uptake can mitigate ON-associated cytotoxicity and genotoxicity.
