Liver-specific inhibition of acyl-coenzyme a:cholesterol acyltransferase 2 with antisense oligonucleotides limits atherosclerosis development in apolipoprotein B100-only low-density lipoprotein receptor-/- mice.

OBJECTIVE: The purpose of this study was to determine the effects of liver-specific inhibition of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) on the development of hypercholesterolemia and atherosclerosis in mice. METHODS AND RESULTS: Apolipoprotein B100-only low-density lipoprotein (LDL) receptor-/- mice were given saline, a nontargeting control antisense oligonucleotide (ASO), or ASOs targeting ACAT2 biweekly for a period spanning 16 weeks. Mice treated with ACAT2 targeting ASOs had liver-specific reduction in ACAT2 mRNA, yet intestinal ACAT2 and cholesterol absorption was left undisturbed. ASO-mediated knockdown of ACAT2 resulted in reduction of total plasma cholesterol, increased levels of plasma triglyceride, and a shift in LDL cholesteryl ester (CE) fatty acid composition from mainly saturated and monounsaturated to polyunsaturated fatty acid enrichment. Furthermore, the liver-specific depletion of ACAT2 resulted in protection against diet-induced hypercholesterolemia and aortic CE deposition. This is the first demonstration that specific pharmacological inhibition of ACAT2, without affecting ACAT1, is atheroprotective. CONCLUSIONS: Hepatic ACAT2 plays a critical role in driving the production of atherogenic lipoproteins, and therapeutic interventions, such as the ACAT2-specific ASOs used here, which reduce acyltransferase 2 (ACAT2) function in the liver without affecting ACAT1, may provide clinical benefit for cardiovascular disease prevention.

Cross-species comparison of in vivo PK/PD relationships for second-generation antisense oligonucleotides targeting apolipoprotein B-100.

The in vivo pharmacokinetics/pharmacodynamics of 2'-O-(2-methoxyethyl) (2'-MOE) modified antisense oligonucleotides (ASOs), targeting apolipoprotein B-100 (apoB-100), were characterized in multiple species. The species-specific apoB antisense inhibitors demonstrated target apoB mRNA reduction in a drug concentration and time-dependent fashion in mice, monkeys, and humans. Consistent with the concentration-dependent decreases in liver apoB mRNA, reductions in serum apoB, and LDL-C, and total cholesterol were concurrently observed in animal models and humans. Additionally, the long duration of effect after cessation of dosing correlated well with the elimination half-life of 2'-MOE modified apoB ASOs studied in mice (t(1/2) congruent with 20 days) and humans (t(1/2) congruent with 30 days) following parental administrations. The plasma concentrations of ISIS 301012, observed in the terminal elimination phase of both mice and monkeys were in equilibrium with liver. The partition ratios between liver and plasma were similar, approximately 6000:1, across species, and thus provide a surrogate for tissue exposure in humans. Using an inhibitory E(max) model, the ASO liver EC(50s) were 101+/-32, 119+/-15, and 300+/-191 microg/g of ASO in high-fat-fed (HF) mice, transgenic mice containing the human apoB transgene, and monkeys, respectively. The estimated liver EC(50) in man, extrapolated from trough plasma exposure, was 81+/-122 microg/g. Therefore, extraordinary consistency of the exposure-response relationship for the apoB antisense inhibitor was observed across species, including human. The cross-species PK/PD relationships provide confidence in the use of pharmacology animal models to predict human dosing for second-generation ASOs targeting the liver.

Antisense inhibition of proprotein convertase subtilisin/kexin type 9 reduces serum LDL in hyperlipidemic mice.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of a family of proteases that is thought to promote the degradation of the low density lipoprotein receptor (LDLR) through an as yet undefined mechanism. We developed second generation antisense oligonucleotide (ASO) inhibitors targeting murine PCSK9 to determine their potential as lipid-lowering agents. Administration of a PCSK9 ASO to high fat-fed mice for 6 weeks reduced total cholesterol and LDL by 53% and 38%, respectively. Moreover, inhibition of PCSK9 expression resulted in a 2-fold increase in hepatic LDLR protein levels. This phenotype closely resembles that reported previously in Pcsk9-deficient mice. The absence of cholesterol lowering in Ldlr-deficient mice effectively demonstrated a critical role for this receptor in mediating the lipid-lowering effects of PCSK9 inhibition. Antisense inhibition of PCSK9 is an attractive and novel therapeutic approach for treating hypercholesterolemia in human.

Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2'-O-methoxyethyl-modified oligonucleotide.

A number of proinflammatory cytokines, including IL-1beta, signal through the adaptor protein MyD88. This signaling leads to phosphorylation of IL-1R-associated kinase-1 (IRAK-1) and, ultimately, activation of the NF-kappaB transcription factor. A splice variant of MyD88 (MyD88(S)), which lacks the ability to couple IRAK-1 to NF-kappaB, has been described. A chemically modified antisense oligonucleotide (ASO) that alters the splicing ratio of MyD88 to MyD88(S) in both cell culture and in animals has been identified. The ASO (ISIS 337846) binds to exon II donor sites in the MyD88 pre-mRNA. By manipulating levels of MyD88 splicing, proinflammatory signaling through the IL-1R has been shown to be diminished, both in cell culture and in mouse liver. To our knowledge, this represents the first example of modulation of RNA splicing of an endogenous gene target in animals after systemic ASO dosing and suggests that this mechanism may be useful as a novel modulator of inflammatory stimuli.

An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis.

High levels of plasma apolipoprotein B-100 (apoB-100), the principal apolipoprotein of LDL, are associated with cardiovascular disease. We hypothesized that suppression of apoB-100 mRNA by an antisense oligonucleotide (ASO) would reduce LDL cholesterol (LDL-C). Because most of the plasma apoB is made in the liver, and antisense drugs distribute to that organ, we tested the effects of a mouse-specific apoB-100 ASO in several mouse models of hyperlipidemia, including C57BL/6 mice fed a high-fat diet, Apoe-deficient mice, and Ldlr-deficient mice. The lead apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and serum apoB-100 levels in a dose- and time-dependent manner. Consistent with those findings, total cholesterol and LDL-C decreased by 25-55% and 40-88%, respectively. Unlike small-molecule inhibitors of microsomal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis and did not affect dietary fat absorption or elevate plasma transaminase levels. These findings, as well as those derived from interim phase I data with a human apoB-100 antisense drug, suggest that antisense inhibition of this target may be a safe and effective approach for the treatment of humans with hyperlipidemia.