Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates - PubMed (original) (raw)

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

Ellen Marie Straarup et al. Nucleic Acids Res. 2010 Nov.

Abstract

The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

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Figures

Figure 1.

Mismatch specificity in mice. Mice were administered i.v. injections of saline, the 12-, 14- and 16-mer and their respective one mismatch versions at 2.5, 5 and 25 mg/kg, respectively on three successive days and sacrificed 24 h after last dose. Kidneys were analyzed for apoB mRNA expression normalized to GAPDH (in percent of saline control group). Data represent mean values ± SD, n = 5. MM indicates mismatch and PM perfect match. Δ_T_m values represent the difference in _T_m between PM and MM oligonucleotides measured against RNA complementary to the perfect match oligonucleotides.

Figure 2.

Quantitative whole-body autoradiography. Tissue distribution in mice of two 35S-labelled oligonucleotides, the 12- and 16-mer (A and B, respectively), administered i.v. Individual mice were sacrificed at 24 h post-administration, snap-frozen and sectioned for whole-body autoradiography. Following exposure the imaging plates were scanned, tissues and organs intensity were quantified by image analyses. (C) Area under the curve values normalized to the specific radioactivity of the dose levels of 16-mer (dark bars) and 12-mer (white bars).

Figure 3.

Single dose in mice. Mice were administered a single i.v. injection of 5, 10, 15, 20 and 25 mg/kg of the 13-mer and sacrificed 24 and 48 h after dosing. (A) The 13-mer oligonucleotide content was measured in liver and presented as function of dose level at 24 h (crosses) and 48 h (boxes) after dosing. The linear correlation (_R_2 = 0.88) showed oligonucleotide content of ∼0.7 µg oligonucleotide/g liver for each 1 mg/kg administered. (B) Liver apoB mRNA expression (crosses) and serum total cholesterol (circles) were measured for all dose levels 24 h (solid line) and 48 h (dashed line) after dosing. Data are expressed as percentage of respectively, apoB mRNA and serum total cholesterol in saline control and presented as mean values ± SD, n = 5/dose level and time point. The apoB mRNA expression was normalized to the expression of GAPDH.

Figure 4.

Duration of effect in mice. Mice were administered a single i.v. injection of saline (diamonds) or 13-mer at 0.5 (white boxes), 1 (dark boxes), 2.5 (crosses) and 5 mg/kg (circles). Livers were analyzed for (A) apoB mRNA expression normalized to GAPDH and (B) serum total cholesterol levels (both in percent of control groups) at days 1, 3, 8, 16 and 32 after administration. (Data represent mean values ± SD, n = 5).

Figure 5.

Multiple dosing in mice. Mice were administered i.v. injections weekly (group 1, left panel) or biweekly (group 2, right panel) for 49 days with either saline (diamonds) or the 13-mer at 1 (dark boxes), 2.5 (crosses) or 5 mg/kg (circles) (arrows on bottom figure indicates dosing days). Serum was analyzed for non-HDL cholesterol (mainly VLDL+LDL) (A and C) and HDL cholesterol (B and D) at 0, 14 and 49 days after first administration. (Data represent mean values ± SD, n = 7).

Figure 6.

Multiple dosing in non-human primates. Cynomolgus monkeys were administered i.v. injections of the either saline (diamonds) or the 13-mer at 2 (dark boxes), 8 (crosses) or 32 (circles) mg/kg once a week for 28 days (arrows on figures indicates dosing days). Serum was sampled 10 days prior to first administration (Day−10) and every week from the first administration. LDL cholesterol (A), HDL cholesterol (B), ALT activity (D) in serum relative to saline control group was measured (±SD, n = 10). A subset of animals at the 8 and 32 mg/kg dose levels were kept treatment-free for an additional seven week period (n = 4). ApoB mRNA expression in liver normalized to β-actin and relative to saline control group (C) at the end of the treatment period (Day 29, ±SD, n = 6) and at the end of the treatment-free period (Day 77, ±SD, n = 4).

Figure 7.

Oligonucleotide content in liver relative to apoB levels in cynomolgus monkeys. The liver tissue content of the 13-mer was measured in all samples from the multiple dosing study in cynomolgus monkeys after the end of the treatment period (Day 29). Data are presented relative to apoB mRNA expression in liver (A) and serum apoB protein levels (B). Best estimated _E_max fit curves are plotted with _R_2 = 0.88 and _R_2 = 0.90 for mRNA and protein levels, respectively.

References

1. Seth PP, Siwkowski A, Allerson CR, Vasquez G, Lee S, Prakash TP, Wancewicz EV, Witchell D, Swayze EE. Short antisense oligonucleotides with novel 2′-4′ conformationaly restricted nucleoside analogues show improved potency without increased toxicity in animals. J. Med. Chem. 2009;52:10–13. - PubMed
1. Swayze EE, Siwkowski AM, Wancewicz EV, Migawa MT, Wyrzykiewicz TK, Hung G, Monia BP, Bennett CF. Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals. Nucleic Acids Res. 2007;35:687–700. - PMC - PubMed
1. Crooke RM, Graham MJ, Lemonidis KM, Whipple CP, Koo S, Perera RJ. An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis. J. Lipid Res. 2005;46:872–878. - PubMed
1. Nishina K, Unno T, Uno Y, Kubodera T, Kanouchi T, Mizusawa H, Yokota T. Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol. Mol. Ther. 2008;16:734–740. - PubMed
1. Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAS. Nature. 2004;432:173–178. - PubMed

Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates - PubMed (original) (raw)