Frank-Kamenetsky Maria, Grefhorst Aldo, Anderson Norma N, Racie Timothy S, Bramlage Birgit, Akinc Akin, Butler David, Charisse Klaus, Dorkin Robert, Fan Yupeng, Gamba-Vitalo Christina, Hadwiger Philipp, Jayaraman Muthusamy, John Matthias, Jayaprakash K Narayanannair, Maier Martin, Nechev Lubomir, Rajeev Kallanthottathil G, Read Timothy, Röhl Ingo, Soutschek Jürgen, Tan Pamela, Wong Jamie, Wang Gang, Zimmermann Tracy, de Fougerolles Antonin, Vornlocher Hans-Peter, Langer Robert, Anderson Daniel G, Manoharan Muthiah, Koteliansky Victor, Horton Jay D, Fitzgerald Kevin
Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11915-20. doi: 10.1073/pnas.0805434105. Epub 2008 Aug 11.
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.
前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK9）调节低密度脂蛋白受体（LDLR）的蛋白水平和功能。PCSK9缺失会增加肝脏中的LDLR水平并降低血浆低密度脂蛋白胆固醇（LDLc），而PCSK9活性过高则会降低肝脏LDLR水平并升高血浆LDLc。在此，我们开发了能够靶向小鼠、大鼠、非人类灵长类动物（NHP）和人类PCSK9的活性、跨物种小干扰RNA（siRNA）。对于体内研究，将PCSK9和对照siRNA配制在脂质纳米颗粒（LNP）中。小鼠和大鼠肝脏特异性siRNA沉默PCSK9可使PCSK9 mRNA水平降低50 - 70%。PCSK9转录本的减少与血浆胆固醇浓度降低多达60%相关。使用5'-RACE证明这些效应是由RNAi机制介导的。在表达人类PCSK9的转基因小鼠中，siRNA使人类PCSK9转录本沉默>70%，并显著降低PCSK9血浆蛋白水平。在NHP中，单剂量靶向PCSK9的siRNA导致血浆PCSK9、载脂蛋白B和LDLc迅速、持久且可逆地降低，而对高密度脂蛋白胆固醇（HDLc）或甘油三酯（TGs）没有可测量的影响。单次静脉推注给药后，PCSK9沉默的效应持续3周。这些结果验证了用RNAi疗法靶向PCSK9作为特异性降低LDLc的一种方法，为开发降低PCSK9的药物作为治疗高胆固醇血症的未来策略铺平了道路。
