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. We measured bile acid composition, cholesterol absorption, and plasma cholesterol. In parallel, we measured markers of cholesterol absorption and synthesis in humans with type 1 diabetes treated with ezetimibe and simvastatin in a double-blind crossover study. RESULTS: Mice with hepatic deletion of the insulin receptor showed marked increases in 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol. This phenotype was entirely reversed by hepatic deletion of FoxO1. FoxO1 is inhibited by insulin and required for the production of 12α-hydroxylated bile acids, which promote intestinal cholesterol absorption and suppress hepatic cholesterol synthesis. Knockdown of Cyp8b1 normalized 12α-hydroxylated bile acid levels and completely prevented hypercholesterolemia in mice with hepatic deletion of the insulin receptor (n=5-30), as well as mouse models of type 1 diabetes (n=5-22). In parallel, the cholesterol absorption inhibitor, ezetimibe, normalized cholesterol absorption and low-density lipoprotein cholesterol in patients with type 1 diabetes as well as, or better than, the cholesterol synthesis inhibitor, simvastatin (n=20). CONCLUSIONS: Insulin, by inhibiting FoxO1 in the liver, reduces 12α-hydroxylated bile acids, cholesterol absorption, and plasma cholesterol levels. Thus, type 1 diabetes leads to a unique set of derangements in cholesterol metabolism, with increased absorption rather than synthesis. These derangements are reversed by ezetimibe, but not statins, which are currently the first line of lipid-lowering treatment in type 1 diabetes. Taken together, these data suggest that a personalized approach to lipid lowering in type 1 diabetes may be more effective and highlight the need for further studies specifically in this group of patients.

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. Interest in the technology continues to grow, and the field has been subject to a significant number of reviews. In this review, we focus on the molecular events that result in the effects observed and use recent clinical results involving several different ASOs to exemplify specific molecular mechanisms and specific issues. We conclude with the prospective on the technology.

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. The effect of ANGPTL3 on HDL-C is typically attributed to its function as an inhibitor of the enzyme endothelial lipase. The ability to facilitate reverse cholesterol transport (RCT), the transport of cholesterol from peripheral tissues back to the liver, is a proposed antiatherogenic property of HDL. However, the effect of ANGPTL3 inhibition on RCT remains unclear. Here, we performed a series of dose-response and RCT studies using an Angptl3 antisense oligonucleotide (ASO) in mouse models with varying plasma lipid profiles ranging from moderately to severely hyperlipidemic. Angptl3 ASO-mediated reduction in HDL-C was limited to the model with moderate lipidemia, where the majority of plasma cholesterol was associated with HDL. Surprisingly, regardless of the effect on HDL-C, treatment with the Angptl3 ASO enhanced RCT in all models tested. The observations from the RCT assays were confirmed in HDL clearance studies, where mice treated with the Angptl3 ASO displayed increased plasma clearance and hepatic uptake of labeled HDL. The results from our studies suggest that inhibition of ANGPTL3 not only reduces levels of proatherogenic lipids but also improves HDL-mediated RCT.

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. The GWA of the HMDP identified two significant associations on chromosomes 4 and 10 with hepatic Malat1 ASO concentrations. Stabilin 2 (Stab2) and vesicle associated membrane protein 3 (Vamp3) were identified by cis-eQTL analysis. HMDP strains with lower Stab2 expression and Stab2 KO mice displayed significantly lower PK than strains with higher Stab2 expression and the wild type (WT) animals respectively, confirming the role of Stab2 in regulating hepatic Malat1 ASO uptake. GWA examining ASO efficacy uncovered three loci associated with Malat1 potency: Small Subunit Processome Component (Utp11l) on chromosome 4, Rho associated coiled-coil containing protein kinase 2 (Rock2) and Aci-reductone dioxygenase (Adi1) on chromosome 12. Our results demonstrate the utility of mouse GWAS using the HMDP in detecting genes capable of impacting the uptake of ASOs, and identifies genes critical for the activity of ASOs in vivo.

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 [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. RESULTS: The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. CONCLUSIONS: Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).

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. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.

Antisense Inhibition of Apolipoprotein C-III in Patients with Hypertriglyceridemia.

BACKGROUND: Apolipoprotein C-III (APOC3) is a key regulator of plasma triglyceride levels. Elevated triglyceride levels are associated with a risk of adverse cardiovascular events and pancreatitis. ISIS 304801 is a second-generation antisense inhibitor of APOC3 synthesis. METHODS: We conducted a randomized, double-blind, placebo-controlled, dose-ranging, phase 2 study to evaluate ISIS 304801 in untreated patients with fasting triglyceride levels between 350 mg per deciliter (4.0 mmol per liter) and 2000 mg per deciliter (22.6 mmol per liter) (ISIS 304801 monotherapy cohort), as well as in patients receiving stable fibrate therapy who had fasting triglyceride levels between 225 mg per deciliter (2.5 mmol per liter) and 2000 mg per deciliter (ISIS 304801-fibrate cohort). Eligible patients were randomly assigned to receive either ISIS 304801, at doses ranging from 100 to 300 mg, or placebo, once weekly for 13 weeks. The primary outcome was the percentage change in APOC3 level from baseline. RESULTS: A total of 57 patients were treated in the ISIS 304801 monotherapy cohort (41 received active agent, and 16 received placebo), and 28 patients were treated in the ISIS 304801-fibrate cohort (20 received active agent, and 8 received placebo). The mean (±SD) baseline triglyceride levels in the two cohorts were 581±291 mg per deciliter (6.6±3.3 mmol per liter) and 376±188 mg per deciliter (4.2±2.1 mmol per liter), respectively. Treatment with ISIS 304801 resulted in dose-dependent and prolonged decreases in plasma APOC3 levels when the drug was administered as a single agent (decreases of 40.0±32.0% in the 100-mg group, 63.8±22.3% in the 200-mg group, and 79.6±9.3% in the 300-mg group, vs. an increase of 4.2±41.7% in the placebo group) and when it was administered as an add-on to fibrates (decreases of 60.2±12.5% in the 200-mg group and 70.9±13.0% in the 300-mg group, vs. a decrease of 2.2±25.2% in the placebo group). Concordant reductions of 31.3 to 70.9% were observed in triglyceride levels. No safety concerns were identified in this short-term study. CONCLUSIONS: We found that treatment with ISIS 304801 was associated with significant lowering of triglyceride levels, among patients with a broad range of baseline levels, through selective antisense inhibition of APOC3 synthesis. (Funded by Isis Pharmaceuticals; ClinicalTrials.gov number, NCT01529424.).

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

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.e., double knockout mice) hepatic steatosis was greatly diminished and fibrosis prevented, on both low-fat and high-fat diets. The mechanisms underlying protection include reduced long chain FA uptake, shifts in FA distribution (lipidomic profiling), and metabolic turnover, specifically decreased hepatic 18:2 FA and triglyceride species and a shift in 18:2 FA use for oxidation versus incorporation into newly synthesized triglyceride. Double knockout mice were protected against fasting-induced hepatic steatosis (a model of enhanced exogenous FA delivery) yet developed steatosis upon induction of hepatic de novo lipogenesis with fructose feeding. Mttp-LKO mice, on either the liver FA binding protein null or Apobec-1 null background (i.e., apolipoprotein B100 only) exhibited only subtle increases in endoplasmic reticulum stress, suggesting that an altered unfolded protein response is unlikely to account for the attenuated phenotype in double knockout mice. Acute, antisense-mediated liver FA binding protein knockdown in Mttp-LKO mice also reduced FA uptake, increased oxidation versus incorporation of 18:2 species with complete reversal of hepatic steatosis, increased hepatic injury, and worsened fibrosis. CONCLUSION: Perturbing exogenous hepatic FA use modulates both hepatic steatosis and fibrosis in the setting of hepatic Mttp deletion, adding new insight into the pathophysiological mechanisms and consequences of defective very low-density lipoprotein secretion. (Hepatology 2017;65:836-852).

Apolipoprotein CIII links islet insulin resistance to ß-cell failure in diabetes.

Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.

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. Due to the importance of the LD as a key component of hepatic lipid storage and as a platform for HCV particle assembly, it seems this dynamic subcellular organelle is a gatekeeper in the pathogenesis of viral hepatitis. Here, we hypothesized that core requires the host LD scaffold protein, perilipin (PLIN)3, to induce hepatic steatosis. To test our hypothesis in vivo, we have studied core-induced hepatic steatosis in the absence or presence of antisense oligonucleotide-mediated knockdown of PLIN3. PLIN3 knockdown blunted HCV core-induced steatosis in transgenic mice fed either chow or a moderate fat diet. Collectively, our studies demonstrate that the LD scaffold protein, PLIN3, is essential for HCV core-induced hepatic steatosis and provide new insights into the pathogenesis of HCV.

Insulin Dissociates the Effects of Liver X Receptor on Lipogenesis, Endoplasmic Reticulum Stress, and Inflammation.

Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor liver X receptor (LXR) is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knock down Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knock-out of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation, and ER stress from the negative effects on lipogenesis.

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. In a phase 2 trial (done in 13 study centres in Canada, the Netherlands, Germany, Denmark, and the UK), we assessed the effect of IONIS-APO(a)Rx, an oligonucleotide targeting apolipoprotein(a). Participants with elevated Lp(a) concentrations (125-437 nmol/L in cohort A; ≥438 nmol/L in cohort B) were randomly assigned (in a 1:1 ratio in cohort A and in a 4:1 ratio in cohort B) with an interactive response system to escalating-dose subcutaneous IONIS-APO(a)Rx (100 mg, 200 mg, and then 300 mg, once a week for 4 weeks each) or injections of saline placebo, once a week, for 12 weeks. Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration at day 85 or 99 in the per-protocol population (participants who received more than six doses of study drug) and safety and tolerability in the safety population. In a phase 1/2a first-in-man trial, we assessed the effect of IONIS-APO(a)-LRx, a ligand-conjugated antisense oligonucleotide designed to be highly and selectively taken up by hepatocytes, at the BioPharma Services phase 1 unit (Toronto, ON, Canada). Healthy volunteers (Lp[a] ≥75 nmol/L) were randomly assigned to receive a single dose of 10-120 mg IONIS-APO(a)LRx subcutaneously in an ascending-dose design or placebo (in a 3:1 ratio; single-ascending-dose phase), or multiple doses of 10 mg, 20 mg, or 40 mg IONIS-APO(a)LRx subcutaneously in an ascending-dose design or placebo (in an 8:2 ratio) at day 1, 3, 5, 8, 15, and 22 (multiple-ascending-dose phase). Primary endpoints were mean percentage change in fasting plasma Lp(a) concentration, safety, and tolerability at day 30 in the single-ascending-dose phase and day 36 in the multiple-ascending-dose phase in participants who were randomised and received at least one dose of study drug. In both trials, the randomised allocation sequence was generated by Ionis Biometrics or external vendor with a permuted-block randomisation method. Participants, investigators, sponsor personnel, and clinical research organisation staff who analysed the data were all masked to the treatment assignments. Both trials are registered with ClinicalTrials.gov, numbers NCT02160899 and NCT02414594. FINDINGS: From June 25, 2014, to Nov 18, 2015, we enrolled 64 participants to the phase 2 trial (51 in cohort A and 13 in cohort B). 35 were randomly assigned to IONIS-APO(a)Rx and 29 to placebo. At day 85/99, participants assigned to IONIS-APO(a)Rx had mean Lp(a) reductions of 66·8% (SD 20·6) in cohort A and 71·6% (13·0) in cohort B (both p<0·0001 vs pooled placebo). From April 15, 2015, to Jan 11, 2016, we enrolled 58 healthy volunteers to the phase 1/2a trial of IONIS-APO(a)-LRx. Of 28 participants in the single-ascending-dose phase, three were randomly assigned to 10 mg, three to 20 mg, three to 40 mg, six to 80 mg, six to 120 mg, and seven to placebo. Of 30 participants in the multiple-ascending-dose phase, eight were randomly assigned to 10 mg, eight to 20 mg, eight to 40 mg, and six to placebo. Significant dose-dependent reductions in mean Lp(a) concentrations were noted in all single-dose IONIS-APO(a)-LRx groups at day 30. In the multidose groups, IONIS-APO(a)-LRx resulted in mean reductions in Lp(a) of 66% (SD 21·8) in the 10 mg group, 80% (SD 13·7%) in the 20 mg group, and 92% (6·5) in the 40 mg group (p=0·0007 for all vs placebo) at day 36. Both antisense oligonucleotides were safe. There were two serious adverse events (myocardial infarctions) in the IONIS-APO(a)Rx phase 2 trial, one in the IONIS-APO(a)Rx and one in the placebo group, but neither were thought to be treatment related. 12% of injections with IONIS-APO(a)Rx were associated with injection-site reactions. IONIS-APO(a)-LRx was associated with no injection-site reactions. INTERPRETATION: IONIS-APO(a)-LRx is a novel, tolerable, potent therapy to reduce Lp(a) concentrations. IONIS-APO(a)-LRx might mitigate Lp(a)-mediated cardiovascular risk and is being developed for patients with elevated Lp(a) concentrations with existing cardiovascular disease or calcific aortic valve stenosis. FUNDING: Ionis Pharmaceuticals.

Targeting APOC3 in the familial chylomicronemia syndrome.

The familial chylomicronemia syndrome is a genetic disorder characterized by severe hypertriglyceridemia and recurrent pancreatitis due to a deficiency in lipoprotein lipase (LPL). Currently, there are no effective therapies except for extreme restriction in the consumption of dietary fat. Apolipoprotein C-III (APOC3) is known to inhibit LPL, although there is also evidence that APOC3 increases the level of plasma triglycerides through an LPL-independent mechanism. We administered an inhibitor of APOC3 messenger RNA (mRNA), called ISIS 304801, to treat three patients with the familial chylomicronemia syndrome and triglyceride levels ranging from 1406 to 2083 mg per deciliter (15.9 to 23.5 mmol per liter). After 13 weeks of study-drug administration, plasma APOC3 levels were reduced by 71 to 90% and triglyceride levels by 56 to 86%. During the study, all patients had a triglyceride level of less than 500 mg per deciliter (5.7 mmol per liter) with treatment. These data support the role of APOC3 as a key regulator of LPL-independent pathways of triglyceride metabolism.

Angiotensinogen Exerts Effects Independent of Angiotensin II.

OBJECTIVE: This study determined whether angiotensinogen (AGT) has angiotensin II-independent effects using multiple genetic and pharmacological manipulations. APPROACH AND RESULTS: All study mice were in low-density lipoprotein receptor -/- background and fed a saturated fat-enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor -/- mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat-enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. CONCLUSIONS: AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.

Antisense inhibition of apolipoprotein (a) to lower plasma lipoprotein (a) levels in humans.

Epidemiological, genetic association, and Mendelian randomization studies have provided strong evidence that lipoprotein (a) [Lp(a)] is an independent causal risk factor for CVD, including myocardial infarction, stroke, peripheral arterial disease, and calcific aortic valve stenosis. Lp(a) levels >50 mg/dl are highly prevalent (20% of the general population) and are overrepresented in patients with CVD and aortic stenosis. These data support the notion that Lp(a) should be a target of therapy for CVD event reduction and to reduce progression of aortic stenosis. However, effective therapies to specifically reduce plasma Lp(a) levels are lacking. Recent animal and human studies have shown that Lp(a) can be specifically targeted with second generation antisense oligonucleotides (ASOs) that inhibit apo(a) mRNA translation. In apo(a) transgenic mice, an apo(a) ASO reduced plasma apo(a)/Lp(a) levels and their associated oxidized phospholipid (OxPL) levels by 86 and 93%, respectively. In cynomolgus monkeys, a second generation apo(a) ASO, ISIS-APO(a)Rx, significantly reduced hepatic apo(a) mRNA expression and plasma Lp(a) levels by >80%. Finally, in a phase I study in normal volunteers, ISIS-APO(a)Rx ASO reduced Lp(a) levels and their associated OxPL levels up to 89 and 93%, respectively, with minimal effects on other lipoproteins. ISIS-APO(a)Rx represents the first specific and potent drug in clinical development to lower Lp(a) levels and may be beneficial in reducing CVD events and progression of calcific aortic valve stenosis.

Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study.

BACKGROUND: Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease and calcific aortic valve stenosis. No effective therapies to lower plasma Lp(a) concentrations exist. We have assessed the safety, pharmacokinetics, and pharmacodynamics of ISIS-APO(a)Rx, a second-generation antisense drug designed to reduce the synthesis of apolipoprotein(a) (apo[a]) in the liver. METHODS: In this randomised, double-blind, placebo-controlled, phase 1 study at the PAREXEL Clinical Pharmacology Research Unit (Harrow, Middlesex, UK), we screened for healthy adults aged 18-65 years, with a body-mass index less than 32·0 kg/m(2), and Lp(a) concentration of 25 nmol/L (100 mg/L) or more. Via a randomisation technique, we randomly assigned participants to receive a single subcutaneous injection of ISIS-APO(a)Rx (50 mg, 100 mg, 200 mg, or 400 mg) or placebo (3:1) in the single-dose part of the study or to receive six subcutaneous injections of ISIS-APO(a)Rx (100 mg, 200 mg, or 300 mg, for a total dose exposure of 600 mg, 1200 mg, or 1800 mg) or placebo (4:1) during a 4 week period in the multi-dose part of the study. Participants, investigators, and study staff were masked to the treatment assignment, except for the pharmacist who prepared the ISIS-APO(a)Rx or placebo. The primary efficacy endpoint was the percentage change from baseline in Lp(a) concentration at 30 days in the single-dose cohorts and at 36 days for the multi-dose cohorts. Safety and tolerability was assessed 1 week after last dose and included determination of the incidence, severity, and dose relation of adverse events and changes in laboratory variables, including lipid panel, routine haematology, blood chemistry, urinalysis, coagulation, and complement variables. Other assessments included vital signs, a physical examination, and 12-lead electrocardiograph. This trial is registered with European Clinical Trials Database, number 2012-004909-27. FINDINGS: Between Feb 27, 2013, and July 15, 2013, 47 (23%) of 206 screened volunteers were randomly assigned to receive ISIS-APO(a)Rx as a single-dose or multi-dose of ascending concentrations or placebo. In the single-dose study, we assigned three participants to receive 50 mg ISIS-APO(a)Rx, three participants to receive 100 mg ISIS-APO(a)Rx, three participants to receive 200 mg ISIS-APO(a)Rx, three participants to receive 400 mg ISIS-APO(a)Rx, and four participants to receive placebo. All 16 participants completed treatment and follow-up and were included in the pharmacodynamics, pharmacokinetics, and safety analyses. For the multi-dose study, we assigned eight participants to receive six doses of 100 mg ISIS-APO(a)Rx, nine participants to receive six doses of 200 mg ISIS-APO(a)Rx, eight participants to receive six doses of 300 mg ISIS-APO(a)Rx, and six participants to receive six doses of placebo. Whereas single doses of ISIS-APO(a)Rx (50-400 mg) did not decrease Lp(a) concentrations at day 30, six doses of ISIS-APO(a)Rx (100-300 mg) resulted in dose-dependent, mean percentage decreases in plasma Lp(a) concentration of 39·6% from baseline in the 100 mg group (p=0·005), 59·0% in the 200 mg group (p=0·001), and 77·8% in the 300 mg group (p=0·001). Similar reductions were observed in the amount of oxidized phospholipids associated with apolipoprotein B-100 and apolipoprotein(a). Mild injection site reactions were the most common adverse events. INTERPRETATION: ISIS-APO(a)Rx results in potent, dose-dependent, selective reductions of plasma Lp(a). The safety and tolerability support continued clinical development of ISIS-APO(a)Rx as a potential therapeutic drug to reduce the risk of cardiovascular disease and calcific aortic valve stenosis in patients with elevated Lp(a) concentration. FUNDING: Isis Pharmaceuticals.

Role of Insulin in the Regulation of Proprotein Convertase Subtilisin/Kexin Type 9.

OBJECTIVE: Proprotein convertase subtilisin/kexin type 9 (PCSK9), which binds the low-density lipoprotein receptor and targets it for degradation, has emerged as an important regulator of serum cholesterol levels and cardiovascular disease risk. Although much work is currently focused on developing therapies for inhibiting PCSK9, the endogenous regulation of PCSK9, particularly by insulin, remains unclear. The objective of these studies was to determine the effects of insulin on PCSK9 in vitro and in vivo. APPROACH AND RESULTS: Using rat hepatoma cells and primary rat hepatocytes, we found that insulin increased PCSK9 expression and increased low-density lipoprotein receptor degradation in a PCSK9-dependent manner. In parallel, hepatic Pcsk9 mRNA and plasma PCSK9 protein levels were reduced by 55% to 75% in mice with liver-specific knockout of the insulin receptor; 75% to 88% in mice made insulin-deficient with streptozotocin; and 65% in ob/ob mice treated with antisense oligonucleotides against the insulin receptor. However, antisense oligonucleotide-mediated knockdown of insulin receptor in lean, wild-type mice had little effect. In addition, we found that fasting was able to reduce PCSK9 expression by 80% even in mice that lack hepatic insulin signaling. CONCLUSIONS: Taken together, these data indicate that although insulin induces PCSK9 expression, it is not the sole or even dominant regulator of PCSK9 under all conditions.

Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.

RATIONALE: Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. OBJECTIVE: To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. METHODS AND RESULTS: Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. CONCLUSIONS: Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

Hepatic Mttp deletion reverses gallstone susceptibility in L-Fabp knockout mice.

Previous studies demonstrated that L-Fabp KO mice are more susceptible to lithogenic diet (LD)-induced gallstones because of altered hepatic cholesterol metabolism and increased canalicular cholesterol secretion. Other studies demonstrated that liver-specific deletion of microsomal triglyceride transfer protein (Mttp-LKO) reduced LD-induced gallstone formation by increasing biliary phospholipid secretion. Here we show that mice with combined deletion (i.e., DKO mice) are protected from LD-induced gallstone formation. Following 2 weeks of LD feeding, 73% of WT and 100% of L-Fabp KO mice developed gallstones versus 18% of Mttp-LKO and 23% of DKO mice. This phenotype was recapitulated in both WT and L-Fabp KO mice treated with an Mttp antisense oligonucleotide (M-ASO). Biliary cholesterol secretion was increased in LD-fed L-Fabp KO mice and decreased in DKO mice. However, phospholipid secretion was unchanged in LD-fed Mttp-LKO and DKO mice as well as in M-ASO-treated mice. Expression of the canalicular export pump ABCG5/G8 was reduced in LD-fed DKO mice and in M-ASO-treated L-Fabp KO mice. We conclude that liver-specific Mttp deletion not only eliminates apical lipoprotein secretion from hepatocytes but also attenuates canalicular cholesterol secretion, which in turn decreases LD-induced gallstone susceptibility.

Effects of antisense oligonucleotides against C-reactive protein on the development of atherosclerosis in WHHL rabbits.

Increased plasma levels of C-reactive protein (CRP) are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO) on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.