The success of mRNA-based therapies depends on the availability of a safe and efficient delivery vehicle. Lipid nanoparticles have been identified as a viable option. However, there are concerns whether an acceptable tolerability profile for chronic dosing can be achieved. The efficiency and tolerability of lipid nanoparticles has been attributed to the amino lipid. Therefore, we developed a new series of amino lipids that address this concern. Clear structure-activity relationships were developed that resulted in a new amino lipid that affords efficient mRNA delivery in rodent and primate models with optimal pharmacokinetics. A 1-month toxicology evaluation in rat and non-human primate demonstrated no adverse events with the new lipid nanoparticle system. Mechanistic studies demonstrate that the improved efficiency can be attributed to increased endosomal escape. This effort has resulted in the first example of the ability to safely repeat dose mRNA-containing lipid nanoparticles in non-human primate at therapeutically relevant levels.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels Ϸ2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR ؊/؊ mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.antibody ͉ LDL-C ͉ LDLR ͉ PCSK9 ͉ hypercholesterolemia P roprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated as an important regulator of LDL metabolism (1, 2). Human genetic studies provide strong validation for the role of PCSK9 in modulating LDL cholesterol (LDL-C) levels and the incidence of coronary heart disease (CHD) in man. Gain-of-function (GOF) mutations in the PCSK9 gene are associated with elevated serum LDL-C levels (Ͼ300 mg/dL) and premature CHD (3), whereas loss-of-function (LOF) mutations are associated with low serum LDL-C (Յ100 mg/dL) (4). Strikingly, subjects harboring the heterozygous LOF mutations exhibited an 88% reduction in the incidence of CHD over a 15-year period relative to noncarriers of the mutations (5). Moreover, despite a complete loss of PCSK9 and serum LDL-C of Ͻ20 mg/dL, the 2 subjects carrying compound heterozygote LOF mutations appear healthy (6, 7).PCSK9 belongs to the subtilisin family of serine proteases and consists of a prodomain, catalytic domain, and C-terminal V domain (8). Expressed highly in the liver, PCSK9 is secreted after autocatalytic cleavage of its zymogen form (1). The prodomain remains noncovalently associated with the catalytic domain and seems to inhibit further proteolytic enzyme activity (8, 9). Secreted PCSK9 modulates LDL-C levels by posttranslational downregulation of hepatic LDL receptor (LDLR) protein (1). The precise mechanism is unknown, but a direct interaction between repeat A of the LDLR EGF homology domain and the PCSK9 catalytic domain is required (10, 11). Proteolytic cleavage of the LDLR by PCSK9 does not occur (12, 13); rather, the PCSK9:LDLR complex is endocytosed and directed to the endosome/lysosome compartment for degradation (14, 15). Current understanding of the LDLR pathway asserts that apolipoprotein B (apoB) and E (apoE) containing lipoprotein particles endocytosed with the LDLR are transported to the acidic environment of the endosome, where they dissociate from the receptor and are subsequently catabolized in lysosomes, while t...

Atrial natriuretic peptide (ANP) is a cardiac hormone that regulates blood pressure. In cardiomyocytes, the hormone is synthesized as a precursor, proatrial natriuretic peptide (pro-ANP), which is proteolytically converted to active ANP. Corin is a cardiac transmembrane serine protease that has been shown to process pro-ANP in vitro, but its physiological importance had not been established. Here, we show that corin-deficient (Cor ؊/؊ ) mice develop normally during embryogenesis and survive to postnatal life. Cor ؊/؊ mice have elevated levels of pro-ANP but no detectable levels of ANP as compared with WT littermates. Infusion of an active recombinant soluble corin transiently restores pro-ANP conversion, resulting in the release of circulating biologically active ANP. Using radiotelemetry to assess blood pressure, we find that Cor ؊/؊ mice have spontaneous hypertension as compared with WT mice, and it is enhanced after dietary salt loading. Pregnant Cor ؊/؊ mice demonstrate late-gestation proteinuria and enhanced high blood pressure during pregnancy. In addition, Cor ؊/؊ mice exhibit cardiac hypertrophy resulting in a mild decline in cardiac function later in life. Thus, our data establish corin as the physiological pro-ANP convertase and indicate that corin deficiency may contribute to hypertensive heart disease.blood pressure ͉ cardiac hypertrophy ͉ preeclampsia ͉ serine protease A trial natriuretic peptide (ANP) is a peptide hormone synthesized in the heart as an inactive precursor, proatrial natriuretic peptide (pro-ANP), which is stored in the dense granules of cardiomyocytes. In response to volume expansion and pressure overload, pro-ANP is secreted from cardiomyocytes and proteolytically cleaved, converting it to the mature peptide, ANP. In target organs such as kidney and blood vessels, ANP stimulates production of intracellular cGMP in a receptordependent manner, leading to natriuresis, diuresis, and vasodilatation, thereby decreasing blood pressure through reduction of intravascular volume and systemic vascular resistance (for review, see ref. 1). Despite the importance of the ANP-mediated pathway in regulating blood pressure and body fluid homeostasis, the enzyme(s) responsible for pro-ANP conversion remained poorly defined.Corin is a type II transmembrane serine protease highly expressed in the heart (2-4). Cell-based studies have shown that recombinant human corin converts pro-ANP to active ANP in a sequence-specific manner (5, 6). More recently, purified recombinant human corin also has been shown to process pro-ANP (7), indicating that corin is likely the pro-ANP convertase. Because other proteases such as thrombin and kallikrein have also been reported to cleave pro-ANP in vitro (8,9), it remained to be demonstrated whether corin indeed is the physiological pro-ANP convertase.To define the biological role of corin, we generated corindeficient mice (Cor Ϫ/Ϫ ) by homologous recombination. Our results show that pro-ANP conversion was abolished in these mice. Cor Ϫ/Ϫ mice develop hypertension that is...

BackgroundThe cardiovascular complications of cancer therapeutics are the focus of the burgeoning field of cardio‐oncology. A common challenge in this field is the impact of cancer drugs on cardiac repolarization (ie, QT prolongation) and the potential risk for the life‐threatening arrhythmia torsades de pointes. Although QT prolongation is not a perfect marker of arrhythmia risk, this has become a primary safety metric among oncologists. Cardiologists caring for patients receiving cancer treatment should become familiar with the drugs associated with QT prolongation, its incidence, and appropriate management strategies to provide meaningful consultation in this complex clinical scenario.Methods and ResultsIn this article, we performed a systematic review (using Preferred Reporting Items of Systematic Reviews and Meta‐Analyses (PRISMA) guidelines) of commonly used cancer drugs to determine the incidence of QT prolongation and clinically relevant arrhythmias. We calculated summary estimates of the incidence of all and clinically relevant QT prolongation as well as arrhythmias and sudden cardiac death. We then describe strategies to prevent, identify, and manage QT prolongation in patients receiving cancer therapy. We identified a total of 173 relevant publications. The weighted incidence of any corrected QT (QTc) prolongation in our systematic review in patients treated with conventional therapies (eg, anthracyclines) ranged from 0% to 22%, although QTc >500 ms, arrhythmias, or sudden cardiac death was extremely rare. The risk of QTc prolongation with targeted therapies (eg, small molecular tyrosine kinase inhibitors) ranged between 0% and 22.7% with severe prolongation (QTc >500 ms) reported in 0% to 5.2% of the patients. Arrhythmias and sudden cardiac death were rare.ConclusionsOur systematic review demonstrates that there is variability in the incidence of QTc prolongation of various cancer drugs; however, the clinical consequence, as defined by arrhythmias or sudden cardiac death, remains rare.

Matings of mice heterozygous for a protein C (PC) deficient allele, produced by targeted PC gene inactivation, yielded the expected Mendelian distribution of PC genotypes. Pups with a total deficiency of PC (PC Ϫ / Ϫ ), obtained at embryonic day (E) 17.5 and at birth, appeared to develop normally macroscopically, but possessed obvious signs of bleeding and thrombosis and did not survive beyond 24 h after delivery. Microscopic examination of tissues and blood vessels of E17.5 PC Ϫ / Ϫ mice revealed their normal development, but scattered microvascular thrombosis in the brain combined with focal necrosis in the liver was observed. In addition, bleeding was noted in the brain near sites of fibrin deposition. The severity of these pathologies was exaggerated in PC Ϫ / Ϫ neonates. Plasma clottable fibrinogen was not detectable in coagulation assays in PC Ϫ / Ϫ neonatal mice, suggestive of fibrinogen depletion and secondary consumptive coagulopathy. Thus, while total PC deficiency did not affect the anatomic development of the embryo, severe perinatal consumptive coagulopathy occurred in the brain and liver of PC Ϫ / Ϫ mice, suggesting that a total PC deficiency is inconsistent with short-term survival. ( J. Clin. Invest. 1998.