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Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to LDL receptors, leading to their degradation. Genetics studies have shown that loss-of-function mutations in PCSK9 result in reduced plasma LDL cholesterol and decreased risk of coronary heart disease. We aimed to investigate the safety and efficacy of ALN-PCS, a small interfering RNA that inhibits PCSK9 synthesis, in healthy volunteers with raised cholesterol who were not on lipid-lowering treatment. We did a randomised, single-blind, placebo-controlled, phase 1 dose-escalation study in healthy adult volunteers with serum LDL cholesterol of 3·00 mmol/L or higher. Participants were randomly assigned in a 3:1 ratio by computer algorithm to receive one dose of intravenous ALN-PCS (with doses ranging from 0·015 to 0·400 mg/kg) or placebo. The primary endpoint was safety and tolerability of ALN-PCS. Secondary endpoints were the pharmacokinetic characteristics of ALN-PCS and its pharmacodynamic effects on PCSK9 and LDL cholesterol. Study participants were masked to treatment assignment. Analysis was per protocol and we used ANCOVA to analyse pharmacodynamic endpoint data. This trial is registered with ClinicalTrials.gov, number NCT01437059. Of 32 participants, 24 were randomly allocated to receive a single dose of ALN-PCS (0·015 mg/kg [n=3], 0·045 mg/kg [n=3], 0·090 mg/kg [n=3], 0·150 mg/kg [n=3], 0·250 mg/kg [n=6], or 0·400 mg/kg [n=6]) and eight to placebo. The proportions of patients affected by treatment-emergent adverse events were similar in the ALN-PCS and placebo groups (19 [79%] vs seven [88%]). ALN-PCS was rapidly distributed, with peak concentration and area under the curve (0 to last measurement) increasing in a roughly dose-proportional way across the dose range tested. In the group given 0·400 mg/kg of ALN-PCS, treatment resulted in a mean 70% reduction in circulating PCSK9 plasma protein (p<0·0001) and a mean 40% reduction in LDL cholesterol from baseline relative to placebo (p<0·0001). Our results suggest that inhibition of PCSK9 synthesis by RNA interference (RNAi) provides a potentially safe mechanism to reduce LDL cholesterol concentration in healthy individuals with raised cholesterol. These results support the further assessment of ALN-PCS in patients with hypercholesterolaemia, including those being treated with statins. This study is the first to show an RNAi drug being used to affect a clinically validated endpoint (ie, LDL cholesterol) in human beings. Alnylam Pharmaceuticals.

In a phase 1 trial, healthy volunteers were assigned to an RNAi therapeutic inhibitor of PCSK9 or placebo. Single doses of 300 mg or more reduced LDL cholesterol by up to 50%; multiple-dose regimens reduced LDL cholesterol by up to 59%. No serious adverse events were reported. An elevated level of low-density lipoprotein (LDL) cholesterol is a major risk factor for cardiovascular disease. 1 Despite the use of statin therapy, alone or in combination with other lipid-lowering medications, many at-risk patients continue to have elevated levels of LDL cholesterol. 2 – 4 Hence, there is a need for additional treatment options for lowering of the LDL cholesterol level to reduce cardiovascular risk. Proprotein convertase subtilisin–kexin type 9 (PCSK9) is a recently identified but well-validated target for LDL cholesterol–lowering therapy. 5 This serine protease, which is expressed and secreted into the bloodstream predominantly by the liver, binds LDL receptors both intracellularly and . . .

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific. The physical size of the commonly used Cas9 from Streptococcus pyogenes poses challenges for CRISPR-Cas genome editing systems that use the adeno-associated virus as a delivery vehicle; here, smaller Cas9 orthologues are characterized, and Cas9 from Staphylococcus aureus allowed targeting of the cholesterol regulatory gene Pcsk9 in the mouse liver. A small Cas9 gene editor enzyme The RNA-guided endonuclease CRISPR-Cas9 is being widely adopted as the genome-editing tool of choice. However, the physical size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) poses problems for applications that use the adeno-associated virus (AAV) as a delivery vehicle. Feng Zhang and colleagues now characterize six smaller Cas9 orthologues. Focusing on Cas9 from Staphylococcus aureus , they packaged it and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Greater than 40% gene modification was observed within a week of injection, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels.

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Immune cell-derived beta-endorphin (END) and other opioid peptides elicit potent and clinically relevant inhibition of pain (analgesia) in inflamed tissue by activation of peripheral opioid receptors. Pro-opiomelanocortin (POMC) is the polypeptide precursor of END and is processed by prohormone convertases (PCs). This study aims to decipher the processing of POMC in lymphocyte subsets in a rat model of unilateral painful hindpaw inflammation. Lymphocytes, isolated from popliteal lymph nodes, were separated into B-cells, T-cells, T-helper cells and cytotoxic T-cells using magnetic cell sorting, and were examined by polymerase chain reaction, immunofluorescence and radioimmunoassay. At 2 h of inflammation, POMC exon 2–3 mRNA was mostly expressed in B- but not in T-cells. Prohormone convertase 1 (PC1) mRNA and protein were upregulated in B-cells and T-helper cells. Prohormone convertase 2 (PC2) was expressed in T- and B-cells, both in inflamed and non-inflamed lymph nodes. END was expressed in B- but not in T-cells. We conclude that POMC, its processing enzymes and END are predominantly expressed in B-lymphocytes at 2 h of paw inflammation.

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN , PCSK5 , PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant ( p  < 5 × 10 −8 ) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis -eQTL SNP rs4702 for FURIN is strongly associated with both diastolic ( p  = 0.012) and systolic ( p  = 0.035) BP levels, as well as peripheral vascular resistance ( p  = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.

Clinical endpoint studies show that lowering of LDL cholesterol reduces atherosclerotic cardiovascular disease and improves outcomes. Despite the effectiveness of statins in reducing LDL cholesterol, individuals treated with statins remain at substantial residual risk of coronary heart disease. Many individuals at risk of coronary heart disease, predominantly those with high baseline LDL cholesterol and those with intolerable side-effects, do not achieve optimum LDL cholesterol targets as recommended by international guidelines with statin treatment.

The inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) protects a variety of cell types against neuronal apoptosis by binding to apolipoprotein E receptor 2 (ApoER2). The present study aimed to determine the association between PCSK9/ApoER2 signaling and neuronal apoptosis following middle cerebral artery occlusion (MCAO) injury in hyperlipidemic mice. For this purpose, C57BL/6 mice fed with a high-fat diet (HFD) for 6 weeks were exposed to MCAO. Subsequently, PCSK9 was inhibited by a lentiviral vector harboring short-hairpin RNA (shRNA) targeting PCSK9, which was stereotaxically injected into the cerebral cortex of mice. At 48 h post-ischemia, hematoxylineosin staining and a terminal deoxynucleotidyl transferase dUTP nick end labeling assay were performed to determine cerebral tissue injury and apoptosis. PCSK9 and ApoER2 expression levels were assessed by reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and western blotting. The results indicated that hyperlipidemia and increased PCSK9 expression were evident in HFD mice. Cerebral histological injury and neuronal apoptosis, as well as PCSK9 and ApoER2 levels, which were increased upon ischemia in hyperlipidemic mice, were attenuated by PCSK9 shRNA treatment. These protective effects of PCSK9 shRNA interference were associated with decreased neuronal apoptosis and a reduced level of ApoER2 expression in the hippocampus and cortex. The data of the present study demonstrated that the PCSK9 shRNA-mediated anti-apoptotic effect induced by MCAO in hyperlipidemic mice is associated with ApoER2 downregulation, which may be a potential new therapy for stroke treatment in patients with hyperlipidemia.