Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals

Abstract

A series of antisense oligonucleotides (ASOs) containing either 2'-O-methoxyethylribose (MOE) or locked nucleic acid (LNA) modifications were designed to investigate whether LNA antisense oligonucleotides (ASOs) have the potential to improve upon MOE based ASO therapeutics. Some, but not all, LNA containing oligonucleotides increased potency for reducing target mRNA in mouse liver up to 5-fold relative to the corresponding MOE containing ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights and body weights. This toxicity was evident for multiple sequences targeting three different biological targets, as well as in mismatch control sequences having no known mRNA targets. Histopathological evaluation of tissues from LNA treated animals confirmed the hepatocellular involvement. Toxicity was observed as early as 4 days after a single administration. In contrast, the corresponding MOE ASOs showed no evidence for toxicity while maintaining the ability to reduce target mRNA. These studies suggest that while LNA ASOs have the potential to improve potency, they impose a significant risk of hepatotoxicity.

Figure 1

(a) Gap design of current generation of ASO therapeutics having 2′-modified ‘wings’ at the 3′ and 5′ ends flanking a central 2′-deoxy gap region. (b) Structures of MOE and LNA nucleosides.

Figure 2

Reduction of TRADD mRNA in bEND cells after transfection with MOE ASO 1a or LNA ASO 1b (top panel) or MOE ASO 4a or LNA ASO 4b (bottom panel).

Figure 3

Reduction of TRADD mRNA for MOE (a series) and LNA (b series) ASOs. *Not Analyzed: due to severe toxicity at 8 days, the 4.5 μmol/kg dose group of 4b was terminated early.

Figure 4

Plasma transaminase levels for MOE (a series) and LNA (b series) ASOs. ^**Data from 8 days. AST 4020 ± 850, ALT 6470 ± 1450, and severe weight loss led to early termination of 4.5 μmol/kg dose group of 4b.

Figure 5

Histopathology of liver sections from mice treated MOE (a series) and LNA (b series) ASOs at 4.5 μmol/kg twice weekly for 3 weeks. Livers from animals treated with LNA ASOs 1b, 4b and 5b present with significant hepatotoxicities as demonstrated in (A), a routing H&E stain showing profound swollen eosinophilic degeneration, cell death (right arrow) and hyperchromatic nuclei (up arrow) of hepatocytes. The immunohistochemistry reveals that the injured hepatocytes appear with cytoplasmic staining (brown stain cells, up arrows) of cleavage caspase 3 (B), pro-apoptotic protein BAX (C) and M30 (D), a neo-epitope generated in epithelial cells as a result of caspase activation (cleavage). In addition a DNA damage and repair associated protein GADD45β (E) and peroxisome membrane protein PMP70 (Supporting Supplementary Figure S2) were both found to have increased cytoplasmic expression in LNA treated livers. IgG control slides of all four IHC markers were negative (data not shown).

Figure 6

Transaminases (bar graph, left scale) and reduction of TRADD mRNA (points on line graph, right scale) after treatment with the 4-10-4 MOE gapmer 1a or the 2-14-2 LNA gapmer 4b.

Figure 7

Transaminases (bar graph, left scale), and reduction of ApoB mRNA (points on line graph, right scale) after treatment with MOE (a series) and LNA (b series) ASOs.

Figure 8

Transaminases (bar graph, left scale), and reduction of PTEN mRNA (points on line graph, right scale) after treatment with MOE (a series) and LNA (b series) ASOs.