III as an independent risk factor for the development of coro-
nary heart disease (CHD).1 Recent epidemiological studies
and meta-analyses have reaffirmed that elevated triglyceride
levels are associated with CHD and a greater risk of disease
recurrence in patients with stable CHD.2–4 Furthermore, CHD
risk is ameliorated when triglyceride levels are reduced.4–6
In This Issue, see p 1401
Editorial, see p 1405
ypertriglyceridemia has been recognized by the National
Cholesterol Education Program Adult Treatment Panel
Very high plasma triglyceride levels (>5.7 mmol/L [500
mg/dL]) are also associated with pancreatitis and may ac-
count for as much as 10% of all cases of routine acute pan-
creatitis and 50% of gestational pancreatitis.7,8 Although the
risk of pancreatitis is considered significant in any patient with
plasma triglyceride levels >11.3 mmol/L (1000 mg/dL), there
is also an increased probability of developing the disease as-
sociated with even more modest levels.9 The pathogenesis of
pancreatitis is still incompletely understood, but thought to be
related to the proinflammatory state produced when triglyc-
eride are metabolized by pancreatic lipases.8 Interestingly,
elevated triglyceride levels seem to increase the incidence of
pancreatitis, but not the severity of attacks.10
Plasma triglyceride are complex lipids primarily transported
on very low–density lipoprotein (VLDL) particles and
chylomicrons that are synthesized in the liver and intestine,
respectively.11 The plasma triglyceride concentration is a
complex polygenic trait, but a variety of genetic determinants
have been identified, including apolipoprotein C-III (apoC-III),
© 2013 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.111.300367
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. (Circ Res. 2013;112:1479-1490.)
Key Words: antisense oligonucleotides ■ apolipoprotein ■ apolipoprotein C-III ■ clinical trial ■ lipids and
lipoproteins ■ pharmacology ■ triglycerides
Original received October 23, 2012; revision received March 26, 2013; accepted March 28, 2013. In February 2013, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 11.98 days.
From Isis Pharmaceuticals, Carlsbad, CA.
*These authors contributed equally.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.
Correspondence to Mark J. Graham, Isis Pharmaceuticals, 2855 Gazelle Court, Carlsbad, CA 92010. E-mail email@example.com
Antisense Oligonucleotide Inhibition of Apolipoprotein
C-III Reduces Plasma Triglycerides in Rodents,
Nonhuman Primates, and Humans
Mark J. Graham,* Richard G. Lee,* Thomas A. Bell III, Wuxia Fu, Adam E. Mullick,
Veronica J. Alexander, Walter Singleton, Nick Viney, Richard Geary, John Su, Brenda F. Baker,
Jennifer Burkey, Stanley T. Crooke, Rosanne M. Crooke
1480 Circulation Research May 24, 2013
lipoprotein lipase, and a number of other genes.12–14 ApoC-III
genetic variants that enhance apoC-III plasma concentrations
were associated with higher plasma triglyceride and an
increase in the incidence of nonalcoholic fatty liver disease.15
Conversely, variants that suppress apoC-III levels, as observed
in a group of Old Order Amish subjects, were associated with
lower triglyceride levels.16 Similar observations have been
made with regard to lipoprotein lipase genetic mutations.17–20
ApoC-III, a key regulator of plasma triglyceride levels, is a
79-aa glycoprotein, synthesized principally in the liver and, to
a lesser extent, by the intestine.21,22 Multiple apoC-III protein
molecules reside on the surface of apoB-containing lipopro-
teins and high-density lipoproteins (HDLs), a percentage of
which exchange rapidly between these particles. The apoC3
gene is located within the apolipoprotein A-1 and apolipo-
protein A-IV gene cluster on chromosome 11q23.23 ApoC-III
expression is suppressed by insulin and is induced by glucose
via transcriptional regulatory elements in the gene promoter
region.24,25 Activation of peroxisome proliferator–activated
receptor-α also reduces apoC-III expression, accounting in
part for the hypotriglyceridemic action of fibrates.26,27
Elevated plasma apoC-III protein possesses several pro-
atherogenic properties. ApoC-III is a potent inhibitor of the
lipolysis of triglyceride-rich lipoproteins by antagonizing apo-
lipoprotein C-II activation of lipoprotein lipase11,21 and hepatic
lipase, which play an important role in both the conversion of
VLDL to intermediate-density lipoproteins to low-density lipo-
protein (LDL) and in the remodeling of HDL.28,29 It has also
been suggested that apoC-III regulates apoB lipoprotein in
apoB lipoprotein metabolism by promoting intrahepatic VLDL
assembly and secretion,11 reducing triglyceride-rich lipoprotein
clearance,28,30 and increasing the formation of atherogenic small
dense LDL.31 Additionally, the enrichment of HDL with apoC-
III may render a normally protective molecule atherogenic.32
There is also evidence that apoC-III promotes inflammation and
endothelial cell dysfunction by enhancing monocytic cell ad-
hesion via increased vascular cell adhesion molecule-1 expres-
sion.32–34 Finally, apoC-III levels are increased in type 1 diabetic
patients and are also thought to be a cofactor in pancreatic β cell
death.4,35 Taken together, these results support the concept that
apoC-III is a multifunctional protein that not only regulates the
metabolism of triglyceride-rich lipoproteins, but may also con-
tribute to CHD and pathophysiological metabolic states.
In most, but not all, studies plasma apoC-III levels have
been positively associated with CHD risk. In mouse models,
genetic ablation of apoC-III had no effect on atherosclerosis36;
however, overexpression of human apoC-III in the low den-
sity lipoprotein receptor (Ldlr)−/− background significantly in-
creased atherosclerosis.37 In humans, it was recently reported
in a prospective study analysis that the risk for fatal or nonfatal
myocardial infarction was significantly increased in subjects
with apoC-III containing VLDL and LDL.30 Furthermore, the
genetic variants that suppress plasma apoC-III levels in the
Old Order Amish subjects and Ashkenazi Jew populations
also exhibited reduced risk of CHD.16,38
Although a variety of therapeutic agents reduce triglyceride
levels (statins, niacin, fibrates, and ω-3 fatty acids),5,39–43 some
patients still cannot meet their recommended triglyceride goals,
suggesting a need for more effective drugs.2 Therefore, we pos-
tulated that a direct inhibitor of apoC-III, itself an independent
CHD risk factor, might provide therapeutic benefit because of its
broad regulatory effects on triglyceride, triglyceride-rich lipopro-
teins, and HDL particle homeostasis. In this article, we describe
the identification and characterization of a second-generation
antisense drug that selectively reduces apoC-III in transgenic
mice, nonhuman primates, and healthy human volunteers. We
demonstrate the selective dose-dependent reduction of apoC-III
with concomitant triglyceride lowering in multiple preclinical
models and species, and in humans. Importantly, we also show
that apoC-III reduction is well tolerated and is not associated with
increased liver triglyceride accumulation or hepatotoxicity. These
results support the advancement of the human apoC-III drug to
phase 2 investigations in patients with hypertriglyceridemia.
An expanded methods section can be found in the online Data
A series of chimeric 20-mer phosphorothioate antisense oligonucle-
otides (ASOs) containing 2′-O-methoxyethyl groups at positions 1 to 5
and 16 to 20 targeted to murine, rat, and human apoC-III mRNA, as well
as a control ASO, were synthesized and purified on an automated DNA
synthesizer using phosphoramidite chemistry as previously described.44
Preclinical Pharmacology Models
An institutional animal care and use committee approved all procedures
and protocols for the preclinical pharmacology studies. See the expand-
ed online Data Supplement Material section for detailed descriptions
of the ASO sequences, animal strains/models, and of all experiments.
Phase I Clinical Trial in Healthy Human Volunteers
A randomized, placebo-controlled, double-blind, ascending dose,
phase 1 study was conducted in healthy volunteers to evaluate the
safety, pharmacokinetics, and pharmacological effects of the human
apoC-III ASO, ISIS 308401, in humans. The study protocol was
approved by a central institutional review board (Institutional Review
Board Services, Canada) and performed in compliance with the
standards of good clinical practice and the Declaration of Helsinki
in its revised edition.45 See Online Figure I for diagrams of the single
and multiple dose cohort schedules, Online Figure II for the flow
of participants through the study, and Online Table I for baseline
characteristics of subjects assigned to the multiple dose cohorts.
Identification of ISIS 304801 (Human ApoC-III
ASO) and Rodent-Specific ApoC-III ASOs
The apoC3 gene, which is conserved in eukaryotes, is
≈500 base pairs in length, containing 3 introns and 4 ex-
ons. The human, rhesus monkey, and cynomolgus monkey
Nonstandard Abbreviations and Acronyms
area under the curve
cholesteryl ester transfer protein
coronary heart disease
very low–density lipoprotein
Graham et al Antisense Inhibition of ApoC-III 1489
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What Is Known?
•? Elevated triglycerides are an independent risk factor for cardiovas-
cular disease, and very high triglyceride levels (>5.7 mmol/L) are
associated with an enhanced risk of pancreatitis.
•? Current therapeutic agents for the treatment of very high triglyceride
levels are limited.
•? Apolipoprotein C-III (apoC-III) is synthesized principally in the liver,
and it regulates serum triglyceride levels.
•? Loss-of-function variants of apoC-III in a group of Old Order
Amish are associated with lower serum triglyceride levels and are
What New Information Does This Article Contribute?
•? ApoC-III biosynthesis could be selectively inhibited by antisense
•? ApoC-III antisense oligonucleotide treatment produces consistent
and significant reductions in serum apoC-III and triglyceride levels in
rodents, nonhuman primates, and man.
•? ApoC-III antisense oligonucleotide drugs are well tolerated in
preclinical models and in a clinical setting, with no evidence of
Individuals with very high triglyceride (>5.7 mmol/L) and apoC-
III levels are at increased risk for developing cardiovascular dis-
ease, metabolic syndrome, diabetes mellitus, and pancreatitis.
Although several therapeutic agents affect triglyceride levels,
there is an unmet need for more effective therapies. Given the
importance of apoC-III in the regulation of triglyceride homeosta-
sis, we developed antisense inhibitors to demonstrate that reduc-
tion of apoC-III could produce therapeutic benefit. In preclinical
rodent and nonhuman primate models and, most importantly, in
man, we demonstrate that selective inhibition of apoC-III is well
tolerated, and that it results in significant, prolonged reductions in
apoC-III and triglyceride levels, enhanced postprandial triglycer-
ide clearance. The human apoC-III antisense drug may be used as
a monotherapy or in combination with other agents, for example,
fibrates. Drug–drug interactions are not anticipated because anti-
sense oligonucleotides are metabolized by nucleases rather than
by metabolic pathways used by traditional small molecules, such
as the cytochrome P450 system. These findings support clinical
evaluation of this apoC-III drug in subjects with severely elevated
triglyceride levels and type 2 diabetics with moderately elevated
triglyceride levels and uncontrolled glucose levels.
Novelty and Significance