Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides

Abstract

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.

Figure 1.

Nucleobase modification strategy to reduce LNA gapmer hepatotoxicity. (A) Position of chemical modification of the nucleobase. The 5- or 2-position of pyrimidine and 8- or 7-position of purine were selected for chemical modifications. (B) Nucleobase derivatives used in the study. Each color indicates a different nucleobase modification.

Figure 2.

Effects of nucleobase modification on TS1-ASO hepatotoxicity and gene expression. (A) Introduction of a modified nucleobase on the TS-ASO. A cytosine, thymine, or guanine nucleobase in the gap region was replaced with a chemically modified analog. (B) Hepatotoxicity of the TS1-ASO series. Left panel: Serum AST and ALT levels. C57BL/6J mice were intravenously injected 10 mg/kg TS1-T4-5 or TS1-G8-6, or 20 mg/kg other TS1-ASO series. Ninety-six hours post injection, serum AST and ALT levels were measured. ^‡The mice were euthanized due to severe physical toxicity within 96 h post administration. ^§The mice were intravenously injected at a dose of 10 mg/kg. Results are expressed as mean ± SE (n = 4). Asterisks indicate a significant difference compared with the control group (**P < 0.01, *P < 0.05). Daggers indicate a significant difference compared with the TS1-ASO-treated group (^††P < 0.01, ^†P < 0.05). Right panel: Schematic illustration of the parent TS1-ASO and TS1-ASO analogs with nucleobase modifications in the gap region. (C) Scatter plots from microarray analysis of the cells treated with TS1-ASO, TS1-C1-7, TS1-T1-5, and TS1-G1-6. The multiplied fluorescence intensity of control group is shown on the horizontal axis, and the proportion of change in gene expression as the results of introduction of each ASO (expressed logarithmically) is shown on the vertical axis. d: Distance; The total number of mismatches, insertions, or deletions between the ASO and complementary RNA sequences. Red dots: d= 0 genes, which have perfect complementarity. Orange dots: d= 1 genes. Light green dots: d= 2 genes. Gray dots: d≧3 genes. (D) The relationship between hepatotoxicity (AST and ALT values) and the index of overall down-regulation. The levels of serum AST and ALT in mice treated with TS1-ASO or nucleobase-modified TS1-ASOs are shown on the vertical axis. The index of overall changes for down-regulated genes classified up to d = 2 in the cells treated with TS1-ASO or nucleobase-modified TS1-ASOs is shown on the horizontal axis. The index was quantified by taking the logarithm of the ratio of gene expression changes and calculating the sum of the absolute value of it (see a conceptual diagram of the index in Supplementary Figure S4). Blue plot indicates TS1-ASO, and each color indicates each TS1-ASO analogue with nucleobase modification.

Figure 3.

Effects of nucleobase modification on TS6-ASO hepatotoxicity and on-target activity. Hepatotoxicity and on-target activity of TS6-ASO series. Left panel: Serum AST and ALT levels. Middle panel: Schematic of the parent TS6-ASO and TS6-ASO analogs with nucleobase modification in the gap region. Right panel: On-target activity. C57BL/6J mice were intravenously injected 20 mg/kg TS6-ASO series. Ninety-six hours post injection, serum AST and ALT levels and Pcsk9 mRNA level were measured. Results are expressed as mean ± SE (n = 4). Asterisks indicate a significant difference compared with the control group (**P < 0.01). Daggers indicate a significant difference compared with the TS6-ASO-treated group (^††P < 0.01).

Figure 4.

Effect of multiple nucleobase modifications on the hepatotoxicity of LNA gapmer ASOs. (A) Hepatotoxicity of the TS1-ASO series. Left panel: Serum AST and ALT levels. Right panel: Schematic of the parent TS1-ASO and TS1-ASO analogs with a single or multiple nucleobase modifications in the gap region. (B) Hepatotoxicity and on-target activity of the TS6-ASO series. Left panel: Serum AST and ALT levels. Middle panel: Schematic of the parent TS1-ASO or TS6-ASO analogs with single or multiple nucleobase modifications in the gap region. Right panel: On-target activity. C57BL/6J mice were intravenously injected 20 mg/kg TS1-ASO series or TS6-ASO series. Ninety-six hours post injection, serum AST and ALT levels and Pcsk9 mRNA level (TS6-ASO target) were measured. Results are expressed as mean ± SE (n = 4). Asterisks indicate a significant difference compared with the control group (**P < 0.01). Daggers indicate a significant difference compared with the TS1-ASO-treated or TS6-ASO-treated group (^††P < 0.01, ^†P < 0.05).

Figure 5.

The relationship between hepatotoxicity (AST values) and T_m values of TS-ASOs or nucleobase-modified TS-ASO. The serum AST levels are shown on the horizontal axis and the T_m values of the duplexes formed between TS-ASOs or nucleobase-modified TS-ASOs and their complementary RNAs are shown on the vertical axis. The red dots indicate TS-ASOs. The blue dots indicate nucleobase-modified TS-ASOs. The arrows indicate nucleobase-modified TS-ASO which decreased the T_m values by >5°C compared with the parent TS-ASO.