Interventional hepatic apoC-III knockdown improves atherosclerotic plaque stability and remodeling by triglyceride lowering.

Apolipoprotein C-III (apoC-III) is a critical regulator of triglyceride metabolism and correlates positively with hypertriglyceridemia and cardiovascular disease (CVD). It remains unclear if therapeutic apoC-III lowering reduces CVD risk and if the CVD correlation depends on the lipid-lowering or antiinflammatory properties. We determined the impact of interventional apoC-III lowering on atherogenesis using an apoC-III antisense oligonucleotide (ASO) in 2 hypertriglyceridemic mouse models where the intervention lowers plasma triglycerides and in a third lipid-refractory model.

Insulin Prevents Hypercholesterolemia by Suppressing 12α-Hydroxylated Bile Acids.

Background: The risk of cardiovascular disease in type 1 diabetes remains extremely high, despite marked advances in blood glucose control and even the widespread use of cholesterol synthesis inhibitors. Thus, a deeper understanding of insulin regulation of cholesterol metabolism, and its disruption in type 1 diabetes, could reveal better treatment strategies.

Methods: To define the mechanisms by which insulin controls plasma cholesterol levels, we knocked down the insulin receptor, FoxO1, and the key bile acid synthesis enzyme, CYP8B1.

Antisense oligonucleotide-mediated inhibition of angiopoietin-like protein 3 increases reverse cholesterol transport in mice.

Supported by an abundance of experimental and genetic evidence, angiopoietin-like protein 3 (ANGPTL3) has emerged as a promising therapeutic target for cardiovascular disease. ANGPTL3 is primarily produced by the liver and is a potent modulator of plasma lipids and lipoproteins. Experimental models and subjects with loss-of-function Angptl3 mutations typically present with lower levels of HDL-C than noncarriers.

Antisense technology: an overview and prospectus.

Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Nine single-stranded antisense oligonucleotide (ASO) drugs representing four chemical classes, two mechanisms of action and four routes of administration have been approved for commercial use, including the first RNA-targeted drug to be a major commercial success, nusinersen. Although all the approved drugs are for use in patients with rare diseases, many of the ASOs in late- and middle-stage clinical development are intended to treat patients with very common diseases.

Antisense technology: A review.

Antisense technology is beginning to deliver on the broad promise of the technology. Ten RNA-targeted drugs including eight single-strand antisense drugs (ASOs) and two double-strand ASOs (siRNAs) have now been approved for commercial use, and the ASOs in phase 2/3 trials are innovative, delivered by multiple routes of administration and focused on both rare and common diseases. In fact, two ASOs are used in cardiovascular outcome studies and several others in very large trials.

Antisense drug discovery and development technology considered in a pharmacological context.

When coined, the term "antisense" included oligonucleotides of any structure, with any chemical modification and designed to work through any post-RNA hybridization mechanism. However, in practice the term "antisense" has been used to describe single stranded oligonucleotides (ss ASOs) designed to hybridize to RNAswhile the term "siRNA" has come to mean double stranded oligonucleotides designed to activate Ago2. However, the two approaches share many common features.

Phosphatidylinositol-(4,5)-Bisphosphate Regulates Plasma Cholesterol Through LDL (Low-Density Lipoprotein) Receptor Lysosomal Degradation.

TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against or a nontargeting control for 3 to 4 weeks.

Trimethylamine N-Oxide Binds and Activates PERK to Promote Metabolic Dysfunction.

The gut-microbe-derived metabolite trimethylamine N-oxide (TMAO) is increased by insulin resistance and associated with several sequelae of metabolic syndrome in humans, including cardiovascular, renal, and neurodegenerative disease. The mechanism by which TMAO promotes disease is unclear. We now reveal the endoplasmic reticulum stress kinase PERK (EIF2AK3) as a receptor for TMAO: TMAO binds to PERK at physiologically relevant concentrations; selectively activates the PERK branch of the unfolded protein response; and induces the transcription factor FoxO1, a key driver of metabolic disease, in a PERK-dependent manner.

Hybrid Mouse Diversity Panel Identifies Genetic Architecture Associated with the Acute Antisense Oligonucleotide-Mediated Inflammatory Response to a 2'--Methoxyethyl Antisense Oligonucleotide.

Although antisense oligonucleotides (ASOs) are well tolerated preclinically and in the clinic, some sequences of ASOs can trigger an inflammatory response leading to B cell and macrophage activation in rodents. This prompted our investigation into the contribution of genetic architecture to the ASO-mediated inflammatory response. Genome-wide association (GWA) and transcriptomic analysis in a hybrid mouse diversity panel (HMDP) were used to identify and validate novel genes involved in the acute and delayed inflammatory response to a single 75 mg/kg dose of an inflammatory 2'--methoxyethyl (2'MOE) modified ASO.

Increased apolipoprotein C3 drives cardiovascular risk in type 1 diabetes.

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study.

Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index.

The molecular mechanisms of toxicity of chemically modified phosphorothioate antisense oligonucleotides (PS-ASOs) are not fully understood. Here, we report that toxic gapmer PS-ASOs containing modifications such as constrained ethyl (cEt), locked nucleic acid (LNA) and 2'-O-methoxyethyl (2'-MOE) bind many cellular proteins with high avidity, altering their function, localization and stability. We show that RNase H1-dependent delocalization of paraspeckle proteins to nucleoli is an early event in PS-ASO toxicity, followed by nucleolar stress, p53 activation and apoptotic cell death.

Antisense oligonucleotides targeting angiotensinogen: insights from animal studies.

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

Mouse genome-wide association studies and systems genetics uncover the genetic architecture associated with hepatic pharmacokinetic and pharmacodynamic properties of a constrained ethyl antisense oligonucleotide targeting Malat1.

Antisense oligonucleotides (ASOs) have demonstrated variation of efficacy in patient populations. This has prompted our investigation into the contribution of genetic architecture to ASO pharmacokinetics (PK) and pharmacodynamics (PD). Genome wide association (GWA) and transcriptomic analysis in a hybrid mouse diversity panel (HMDP) were used to identify and validate novel genes involved in the uptake and efficacy of a single dose of a Malat1 constrained ethyl (cEt) modified ASO.

Characterization of the Activity and Distribution of a 2'-O-Methoxyethyl-Modified Antisense Oligonucleotide in Models of Acute and Chronic Kidney Disease.

To determine if the pharmacokinetics and pharmacodynamics of gapmer antisense oligonucleotides (ASOs), containing phosphorothioate backbones and 2'-O-methoxyethyl RNA modifications (2'-MOE ASOs), can be altered by renal disease, a series of experiments were performed in models of chronic kidney disease (CKD) and acute kidney injury (AKI). In an adenine diet model of CKD, 2'-MOE ASO activity in the whole kidney was preserved and the reduction in target RNA was sustained for 2-4 weeks postdose. Additionally, 2'-MOE ASO distribution within the kidney was altered in mice with CKD, in that ASO delivery to cortical regions with tubular damage was reduced while distribution to the medulla was increased.

Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs.

Blood Pressure Lowering and Safety Improvements With Liver Angiotensinogen Inhibition in Models of Hypertension and Kidney Injury.

Uncontrolled hypertension is an important contributor to cardiovascular disease. Despite the armamentarium of antihypertensive treatments, there remains a need for novel agents effective in individuals who cannot reach acceptable blood pressure levels. Inhibitors targeting the renin-angiotensin-aldosterone system (RAAS) are widely used but may not optimally inhibit RAAS and demonstrate an acceptable safety profile.

The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue.

Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes.

Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides.

Background: Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins.

Methods: We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.

The lipid droplet-associated protein perilipin 3 facilitates hepatitis C virus-driven hepatic steatosis.

Hepatitis C virus (HCV) is an enveloped RNA virus responsible for 170 million cases of viral hepatitis worldwide. Over 50% of chronically infected HCV patients develop hepatic steatosis, and steatosis can be induced by expression of HCV core protein (core) alone. Additionally, core must associate with cytoplasmic lipid droplets (LDs) for steatosis development and viral particle assembly.

Prevention of hepatic fibrosis with liver microsomal triglyceride transfer protein deletion in liver fatty acid binding protein null mice.

Unlabelled: Blocking hepatic very low-density lipoprotein secretion through genetic or pharmacologic inhibition of microsomal triglyceride transfer protein (Mttp) causes hepatic steatosis, yet the risks for developing hepatic fibrosis are poorly understood. We report that liver-specific Mttp knockout mice (Mttp-LKO) exhibit both steatosis and fibrosis, which is exacerbated by a high-transfat/fructose diet. When crossed into germline liver fatty acid (FA) binding protein null mice (Mttp-LKO, i.

Antisense oligonucleotides targeting apolipoprotein(a) in people with raised lipoprotein(a): two randomised, double-blind, placebo-controlled, dose-ranging trials.

Background: Elevated lipoprotein(a) (Lp[a]) is a highly prevalent (around 20% of people) genetic risk factor for cardiovascular disease and calcific aortic valve stenosis, but no approved specific therapy exists to substantially lower Lp(a) concentrations. We aimed to assess the efficacy, safety, and tolerability of two unique antisense oligonucleotides designed to lower Lp(a) concentrations.

Methods: We did two randomised, double-blind, placebo-controlled trials.

ApoC-III inhibits clearance of triglyceride-rich lipoproteins through LDL family receptors.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease, and plasma triglycerides (TGs) correlate strongly with plasma apolipoprotein C-III (ApoC-III) levels. Antisense oligonucleotides (ASOs) for ApoC-III reduce plasma TGs in primates and mice, but the underlying mechanism of action remains controversial. We determined that a murine-specific ApoC-III-targeting ASO reduces fasting TG levels through a mechanism that is dependent on low-density lipoprotein receptors (LDLRs) and LDLR-related protein 1 (LRP1).