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

doi:10.1073/pnas.0805434105

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

*Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142;
Departments of ^†Molecular Genetics and
^‖Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390;
^‡Roche Kulmbach GmbH, Fritz-Hornschuch-Strasse 9, 95326 Kulmbach, Germany; and
^¶David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139

Contributed by Robert Langer, June 6, 2008 (received for review May 7, 2008)

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.

Footnotes

^§To whom correspondence may be addressed. E-mail: kfitzgerald{at}alnylam.com or rlanger{at}mit.edu
Author contributions: M.F.-K., H.-P.V., M. Manoharan, V.K., J.D.H., and K.F. designed the research; M.F.-K., A.G., N.N.A., T.S.R., B.B., M. John, J.W., and K.F. performed the research; A.A., D.B., K.C., R.D., Y.F., P.H., M. Jayaraman, K.N.J., M. Maier, L.N., K.G.R., T.R., I.R., J.S., P.T., G.W., T.Z., A.d.F., R.L., and D.G.A. contributed new reagents/analytical tools; M.F.-K., C.G.-V., J.D.H., and K.F. analyzed the data; and M.F.-K., J.D.H., and K.F. wrote the paper.
Conflict of interest statement: R.L. is a shareholder and member of the Scientific Advisory Board of Alnylam. D.G.A. and J.D.H. are consultants of Alynylam Pharmaceuticals. Alnylam also has a license to certain intellectual property invented at Massachusetts Institute of Technology by Drs. Anderson, Langer, and colleagues. M.F.-K., T.S.R., A.A., D.B., K.C., R.D., Y.F., C.G.-V., M. Jayaraman, K.N.J., M. Maier, L.N., K.G.R., T.R., J.S., J.W., G.W., T.Z., A.d.F., M. Manoharan, V.K., and K.F. are employees of Alnylam Pharmaceuticals. B.B., P.H., M. John, I.R., P.T., and H.-P.V. are employees of Roche Kulmbach.
This article contains supporting information online at www.pnas.org/cgi/content/full/0805434105/DCSupplemental.
Freely available online through the PNAS open access option.