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


RESULTS
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.


CONCLUSIONS
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.
Gupta
Nidhi
N
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
Fisker
Niels
N
Asselin
Marie-Claude
MC
Lindholm
Marie
M
Rosenbohm
Christoph
C
Ørum
Henrik
H
Elmén
Joacim
J
Seidah
Nabil G
NG
Straarup
Ellen Marie
EM
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D020868:Gene Silencing / Q000187:drug effects*; D056945:Hep G2 Cells; D006801:Humans; D007275:Injections, Intravenous; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009841:Oligonucleotides / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*
D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011973:Receptors, LDL; C477371:locked nucleic acid; C472125:PCSK9 protein, human; C472126:Pcsk9 protein, mouse; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases