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

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.

Kinome-wide RNAi screen implicates at least 5 host hepatocyte kinases in Plasmodium sporozoite infection.

Plasmodium sporozoites, the causative agent of malaria, are injected into their vertebrate host through the bite of an infected Anopheles mosquito, homing to the liver where they invade hepatocytes to proliferate and develop into merozoites that, upon reaching the bloodstream, give rise to the clinical phase of infection. To investigate how host cell signal transduction pathways affect hepatocyte infection, we used RNAi to systematically test the entire kinome and associated genes in human Huh7 hepatoma cells for their potential roles during infection by P. berghei sporozoites. The three-phase screen covered 727 genes, which were tested with a total of 2,307 individual siRNAs using an automated microscopy assay to quantify infection rates and qRT-PCR to assess silencing levels. Five protein kinases thereby emerged as top hits, all of which caused significant reductions in infection when silenced by RNAi. Follow-up validation experiments on one of these hits, PKCsigma (PKCzeta), confirmed the physiological relevance of our findings by reproducing the inhibitory effect on P. berghei infection in adult mice treated systemically with liposome-formulated PKCsigma-targeting siRNAs. Additional cell-based analyses using a pseudo-substrate inhibitor of PKCsigma added further RNAi-independent support, indicating a role for host PKCsigma on the invasion of hepatocytes by sporozoites. This study represents the first comprehensive, functional genomics-driven identification of novel host factors involved in Plasmodium sporozoite infection.

Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing.

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.