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A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo.

Gupta N, Fisker N, Asselin MC, Lindholm M, Rosenbohm C, Ørum H, Elmén J, Seidah NG, Straarup EM - PLoS ONE (2010)

Bottom Line: The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days.Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates.The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada.

ABSTRACT

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.

Methodology/principal findings: The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity.

Conclusion/significance: LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

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Related in: MedlinePlus

Intracellular reduction of targeted PCSK9 mRNA by LNA ASO.Two human hepatic cell lines: HepG2 and HuH7 and a mouse insulinoma β-TC3 cell line; were transfected with LNA ASO, at concentrations of 10 and 25 nM. Total RNA was extracted at two different time points- 24 h and 48 h post transfection and QPCR analysis was performed using specific primers. The levels of PCSK9 mRNA were normalized to S14 mRNA for (A) HepG2 and (B) HuH7 cells; and (C) with S16 mRNA for β-TC3 cells.
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pone-0010682-g001: Intracellular reduction of targeted PCSK9 mRNA by LNA ASO.Two human hepatic cell lines: HepG2 and HuH7 and a mouse insulinoma β-TC3 cell line; were transfected with LNA ASO, at concentrations of 10 and 25 nM. Total RNA was extracted at two different time points- 24 h and 48 h post transfection and QPCR analysis was performed using specific primers. The levels of PCSK9 mRNA were normalized to S14 mRNA for (A) HepG2 and (B) HuH7 cells; and (C) with S16 mRNA for β-TC3 cells.

Mentions: As shown in Figure 1, more than 60% reduction in PCSK9 mRNA levels in human HepG2 (panel A; P<0.01), and approximately 50% in HuH7 (panel B; P<0.02) and mouse β-TC3 (panel C; P<0.05) cells were achieved after 24 h using either 10 or 25 nM LNA ASO. The same trend remained at 48 h post-transfection. We did not see any significant LNA-mediated change in the expression of LDLR mRNA in HepG2 (Figure 2A; P<0.01), HuH7 (Figure 2B; P<0.002) or in β-TC3 (Figure 2C; P<0.05) cells at the 48 h time point. Also, following 48h incubation no significant change was observed in the levels of HMGCoAR mRNA in HepG2 cells (Figure 3A; P<0.02) or in β-TC3 cells (Figure 3B; P<0.03).


A locked nucleic acid antisense oligonucleotide (LNA) silences PCSK9 and enhances LDLR expression in vitro and in vivo.

Gupta N, Fisker N, Asselin MC, Lindholm M, Rosenbohm C, Ørum H, Elmén J, Seidah NG, Straarup EM - PLoS ONE (2010)

Intracellular reduction of targeted PCSK9 mRNA by LNA ASO.Two human hepatic cell lines: HepG2 and HuH7 and a mouse insulinoma β-TC3 cell line; were transfected with LNA ASO, at concentrations of 10 and 25 nM. Total RNA was extracted at two different time points- 24 h and 48 h post transfection and QPCR analysis was performed using specific primers. The levels of PCSK9 mRNA were normalized to S14 mRNA for (A) HepG2 and (B) HuH7 cells; and (C) with S16 mRNA for β-TC3 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871785&req=5

pone-0010682-g001: Intracellular reduction of targeted PCSK9 mRNA by LNA ASO.Two human hepatic cell lines: HepG2 and HuH7 and a mouse insulinoma β-TC3 cell line; were transfected with LNA ASO, at concentrations of 10 and 25 nM. Total RNA was extracted at two different time points- 24 h and 48 h post transfection and QPCR analysis was performed using specific primers. The levels of PCSK9 mRNA were normalized to S14 mRNA for (A) HepG2 and (B) HuH7 cells; and (C) with S16 mRNA for β-TC3 cells.
Mentions: As shown in Figure 1, more than 60% reduction in PCSK9 mRNA levels in human HepG2 (panel A; P<0.01), and approximately 50% in HuH7 (panel B; P<0.02) and mouse β-TC3 (panel C; P<0.05) cells were achieved after 24 h using either 10 or 25 nM LNA ASO. The same trend remained at 48 h post-transfection. We did not see any significant LNA-mediated change in the expression of LDLR mRNA in HepG2 (Figure 2A; P<0.01), HuH7 (Figure 2B; P<0.002) or in β-TC3 (Figure 2C; P<0.05) cells at the 48 h time point. Also, following 48h incubation no significant change was observed in the levels of HMGCoAR mRNA in HepG2 cells (Figure 3A; P<0.02) or in β-TC3 cells (Figure 3B; P<0.03).

Bottom Line: The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days.Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates.The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada.

ABSTRACT

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in the etiology of familial hypercholesterolemia (FH) and is also an attractive therapeutic target to reduce low density lipoprotein (LDL) cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR) and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.

Methodology/principal findings: The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA) antisense oligonucleotide (LNA ASO) that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT) levels revealed that long term LNA ASO treatment (7 weeks) does not cause hepatotoxicity.

Conclusion/significance: LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

Show MeSH
Related in: MedlinePlus