Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50 -70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia. plasma PCSK9 ͉ tissue LDLR levels P roprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the mammalian serine proprotein convertase family that typically functions in the proteolytic processing and maturation of secretory proteins (1, 2). PCSK9 was the first family member to be implicated in a dominantly inherited form of hypercholesterolemia (3). Mechanistic studies addressing the function of PCSK9 in mice and humans have demonstrated that overexpression or gain-of-function mutations in PCSK9 reduced low density lipoprotein receptor (LDLR) protein levels in liver, which significantly increased circulating plasma cholesterol both in mice and humans (4). Additional studies showed that the deletion of Pcsk9 in mice resulted in increased LDLR levels, accelerated the clearance of low density lipoprotein cholesterol (LDLc), and reduced circulating cholesterol levels (5). Recently, studies in mice have also shown that lowering PCSK9 transcript levels by antisense oligonucleotides resulted in reduced total cholesterol, LDLc, and HDL cholesterol (HDLc) in blood and increased LDLR levels in liver after 6 weeks of treatment (6). This effect was very similar to that observed in the Pcsk9 Ϫ/Ϫ mice (5). Collectively, these studies have clearly established a role for PCSK9 in cholesterol homeostasis.Validation of PCSK9 as an attractive therapeutic target for the treatment of hypercholesterolemia has come from genetic studies in humans. Cohen et al. (7) first identified loss-of-function mutations in PCSK9...

Lipid-like materials for low-dose, in vivo gene silencing

“…administration should be possible while remaining well within the range of tolerability established in previous experiments. It could be envisioned that the ability to regulate multiple genes may provide a powerful therapeutic approach to diseases in which multiple gene targets have already been identified (14,19). To investigate the feasibility of this approach, siRNA sequences against liver targets of possible therapeutic interest, Factor VII, ApoB, PCSK9, Xbp1, and SORT1, were pooled and formulated with C12-200.…”

RNAi-mediated silencing of hepatic Alas1 effectively prevents and treats the induced acute attacks in acute intermittent porphyria mice

“…To identify a potent Alas1-siRNA, a panel of siRNAs targeting Alas1 was initially screened for their ability to inhibit Alas1 mRNA expression in cultured hepatic cells. The most active compounds were formulated into lipid nanoparticles (LNPs) for efficient hepatic delivery (21)(22)(23) and evaluated for their ability to down-regulate hepatic Alas1 mRNA expression in wild-type mice. The selected Alas1-siRNA was then evaluated in the mouse model for AIP for its effectiveness to prevent or treat an induced acute attack.…”

Squalene monooxygenase – a target for hypercholesterolemic therapy

“…It should be as effective as statins in the reduction of cholesterol de novo synthesis, but devoid of their side effects. The best way to meet these requirements is to inhibit the squalene monooxygenase activity or silence its gene transcription supported by other approaches such as, diet, inhibition of cholesterol and bile acids absorption, reduction of LDL particles formation by antisense oligonucleotides directed against proprotein convertase subtilisin/kexin type 9 (PCSK9) (Frank-Kemenetsky et al , 2008 ;Rizzo , 2010 ) or against apolipoprotein B-100 (apoB) -mipomersen (Crooke et al , 2005 ;Patel and Hegele , 2010 ), increasing number of LDL receptors (enhancers of LDL receptors synthesis and inhibitors of its degradation) (Lagace et al , 2006 ;Grahan et al, 2007;Shan et al , 2008 ) and other cholesterol transporters (ATP-binding cassette proteins) (Quazi and Molday , 2011 ), and raising high-density lipoprotein levels [cholesteryl ester transfer protein (CETP) inhibitors (Barter et al , 2007 ;KuangYuh, et al, 2011 )]. Most of them are still under investigation.…”