Development of a 2',4'-BNA/LNA-based siRNA for Dyslipidemia and Assessment of the Effects of Its Chemical Modifications In Vivo

Abstract

Recent advances in RNA interference (RNAi)-based drug development have partially allowed systemic administration of these agents in vivo with promising therapeutic effects. However, before chemically modified small-interfering RNAs (siRNAs) can be applied clinically, their in vivo effects should be thoroughly assessed. And while many studies have assessed the effects of chemically modified siRNAs in vitro, there has been no comprehensive assessment of their effects in vivo. Here, we aimed to elucidate the effects of administering chemically modified siRNAs in vivo and to propose a 2',4'-bridged nucleic acid (BNA)/locked nucleic acid (LNA)-based siRNA candidate for dyslipidemia. A potentially therapeutic siRNA, siL2PT-1M, was modified with phosphorothioate (PS) and 2',4'-BNA/LNA in its sense strand and with 2'-methoxy (2'-OMe) nucleotides in its immunostimulatory motif; administration of siL2PT-1M resulted in sustained reductions in serum total cholesterol (TC) (24 days) and a concomitant apolipoprotein B (apoB) mRNA reduction in liver without adverse effects. The 2',4'-BNA/LNA modification in the sense strand was greatly augmented the duration of the RNAi effect, whereas cholesterol conjugation shortened the duration. Cholesterol-conjugated immunostimulatory siRNA (isRNA) induced higher serum interferon-α (IFN-α) levels than did nonmodified isRNA, indicating that the immune reaction was facilitated by cholesterol conjugation. Our results indicated that modification of the adenosine residues complementary to the immunostimulatory motif and of central 5'-UG-3' in the sense strand would ameliorate the negative immune response.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.32; published online 18 September 2012.

Figure 1

Structures of chemical modifications used in the study and concentration-dependent RNAi effect of nonmodified or 2′,4′-BNA/LNA modified siRNAs. (a) Structures of the artificial nucleic acid and cholesterol conjugates used in this study. (b) RNAi effect of 2′,4′-BNA/LNA modified siRNAs on apolipoprotein B (apoB) mRNA in NMuLi, mouse hepatic cells. ApoB mRNA expression was normalized to GAPDH expression. Error bar indicates SD values in this experiment.

Figure 2

2′A′-BNA/LNA modification contributed to the nuclease resistance of the siRNAs. Serum stability of siLNAs and nonmodified siApoB-1 in mouse serum was analyzed using incubation times of 30, 60, 120, 360, 720, 1,440, and 2,880 minutes. (a) Samples taken at each time were subjected to native polyacrylamide gel electrophoresis in a 20% TBE gel. Images of polyacrylamide gels stained with SYBR gold are shown. (b) The density of bands representing duplex siRNAs was quantified using Image J software, which is freely available on the Internet. Band intensities were normalized using the band intensity of the first “0 minute” time point.

Figure 3

RNAi effects on apoB mRNA of modified siRNAs based on siLNA-2 and the downstream effects on IFIT-1 mRNA levels. (a) All siRNAs were transfected into NMuLi cells at concentrations of 10 or 100 nmol/l using Lipofectamine RNAiMAX. ApoB mRNA expression was normalized to GAPDH expression, and relative values were calculated using the no-transfection group. (b) Expression of interferon-induced tetratricopeptide repeats 1 (IFIT-1) mRNA was measured as the inflammatory response of the cell; IFIT-1 expression was normalized to GAPDH expression. Error bars indicate SD.

Figure 4

Therapeutic effects and body weight changes in mice receiving an siRNAs. (a) Body appearance, including body weight, of each mouse was observed for a week prior to the 2 days before the injection. Body weight changes in mice treated with an siRNA were indicated as ratio relative to that in the PBS-treated group. (b) Serum total cholesterol levels were indicated as ratio relative to that in the PBS-treated group. (c) ApoB mRNA expression in the liver tissues was normalized to GAPDH expression and was indicated as ratio relative to that in the PBS-treated group. (d) ApoB-100 protein levels in liver tissues were quantified by determining the intensity of the band on a western blot membrane, and these intensities are presented as a ratio relative to the band intensities of samples from the PBS-treated group. All values are the means ±SD of three to five animals.

Figure 5

Changes in serum chemistry after the injection of an siRNAs. Aspartate aminotransaminase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine levels were measured at each time point as indicators of hepatotoxicity and nephrotoxicity. Raw data on (a) AST, (b) ALT, (c) BUN, and (d) Creatinine levels are indicated. All values are the means ±SD of values from three to five animals.

Figure 6

Serum interferon-α (IFN-α) levels and IFN-α-related gene expression. (a) Serum IFN-α levels were measured 6 hours after the injection of each siRNAs using enzyme-linked immunosorbent assay. (b) Interferon-induced tetratricopeptide repeats 1 (IFIT-1) and (c) 2′-5′-oligoadenylate synthetase 1 (OAS-1) mRNA expression in the liver was measured at each time point. IFIT-1 and OAS-1 mRNA expression was normalized to GAPDH expression. All values are the means ±SD of four to five animals.

Figure 7

Liver and kidney tissue damage was assessed histopathologically using H&E staining. (a) Day 2 liver tissues of siNTCtrl, siApoB-1, siL2PT-1, and siL2PTC-1M were indicated as representative. (b) Day 2 kidney tissues of PBS and siApoB-1 were indicated as representative. (c) The number of mice with inflammation in the liver tissue was counted in each group, and the incidence ratio was indicated.

Figure 8

Pharmacological parameters and innate immune responses 2 days after the injection of siApoB-1C or siApoB-1. (a) ApoB mRNA inhibition, apoB-100 protein levels in the liver, and serum TC levels were measured 2 days after the injections. (b) IFN-α levels in serum 6 hours after injection of siApoB-1 and siApoB-1C. (c) Comparison of the effects of siApoB-1 and siApoB-1C on IFN-α-related gene expression. IFIT-1 and OAS-1 mRNA expression was normalized to GAPDH expression. All values are the means ± SD of three to four animals.

Figure 9

Comparison between siL2PT-1-, siL2PTC-1M-, and siL2PT-1M-mediated effects on pharmacological parameters and immune responses on day 2. (a) ApoB mRNA inhibition, apoB-100 protein levels in the liver, and serum TC levels were all measured 2 days after the injection of the siRNAs. (b) IFN-α levels in serum 6 hours after injection of each siRNA. (c) Comparison of the effects of siL2PT-1, siL2PTC-1M and siL2PT-1M on IFN-α-related gene expression. IFIT-1 and OAS-1 mRNA expression was normalized to GAPDH expression. All values are the means ± SD of three to four animals.