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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

Cell viability measurement.HepG2 cells were transfected for 4h with water for mock or 10 and 25 nM LNA ASO using Lipofectamine 2000 and incubated for 48h. The cells were then treated with Alamar blue reagent and analyzed on an Elisa plate reader for cell viability.
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pone-0010682-g006: Cell viability measurement.HepG2 cells were transfected for 4h with water for mock or 10 and 25 nM LNA ASO using Lipofectamine 2000 and incubated for 48h. The cells were then treated with Alamar blue reagent and analyzed on an Elisa plate reader for cell viability.

Mentions: To assess the potential cytotoxicity of the LNA ASO used in this study, cell viability was determined. Cell confluency, a qualitative measure of cell viability based upon cell coverage on the well surface, as judged by microscopy, indicated no visible toxicity at the concentrations of LNA used for transfection. To obtain a more quantitative measure of cell viability, we assayed cell metabolism activity using Alamar blue [36], [37]. LNA ASO was found to be almost non toxic, with more than 85% of the cells being viable after 48h of incubation with 10 and 25 nM LNA ASO (Figure 6).


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)

Cell viability measurement.HepG2 cells were transfected for 4h with water for mock or 10 and 25 nM LNA ASO using Lipofectamine 2000 and incubated for 48h. The cells were then treated with Alamar blue reagent and analyzed on an Elisa plate reader for cell viability.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010682-g006: Cell viability measurement.HepG2 cells were transfected for 4h with water for mock or 10 and 25 nM LNA ASO using Lipofectamine 2000 and incubated for 48h. The cells were then treated with Alamar blue reagent and analyzed on an Elisa plate reader for cell viability.
Mentions: To assess the potential cytotoxicity of the LNA ASO used in this study, cell viability was determined. Cell confluency, a qualitative measure of cell viability based upon cell coverage on the well surface, as judged by microscopy, indicated no visible toxicity at the concentrations of LNA used for transfection. To obtain a more quantitative measure of cell viability, we assayed cell metabolism activity using Alamar blue [36], [37]. LNA ASO was found to be almost non toxic, with more than 85% of the cells being viable after 48h of incubation with 10 and 25 nM LNA ASO (Figure 6).

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