- Email: [email protected]
Genetics for the Identification of Lipid Targets Beyond PCSK9
Accepted Manuscript Genetics for the identification of lipid targets - beyond PCSK9 Linda R. Wang, MD, Robert A. Hegele, MD, FRCPC PII:
S0828-282X(16)31088-1
DOI:
10.1016/j.cjca.2016.11.003
Reference:
CJCA 2300
To appear in:
Canadian Journal of Cardiology
Received Date: 19 September 2016 Revised Date:
6 November 2016
Accepted Date: 7 November 2016
Please cite this article as: Wang LR, Hegele RA, Genetics for the identification of lipid targets - beyond PCSK9, Canadian Journal of Cardiology (2016), doi: 10.1016/j.cjca.2016.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Department of Medicine and Robarts Research Institute, Schulich School of Medicine and Dentistry,
Correspondence: Robert Hegele MD, FRCPC, FACP, FCCS Robarts Research Institute
London, Ontario N6A 5B7
tel: 519-931-5271
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fax: 519-931-5218
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email: [email protected]
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4288A - 1151 Richmond Street North
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Western University, London, Ontario N6A 5B7
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Linda R. Wang MD and Robert A. Hegele MD, FRCPC
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Genetics for the identification of lipid targets - beyond PCSK9
ACCEPTED MANUSCRIPT 2 Abstract From studies of rare families to genome-wide associations in populations, understanding of human genetics has accelerated the search for new drug targets for the prevention of atherosclerotic
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cardiovascular disease. DNA sequencing and genome-wide analyses of DNA markers have illuminated both rare and common variants in genes that regulate lipids and ultimately atherosclerosis risk. A
recent innovative approach called Mendelian randomization can endorse specific genes and variants as
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causative not just for lipid disturbances, but also for clinical cardiovascular end points. This knowledge helps prioritize the candidate genes and proteins in the drug development pipeline. In this review, we
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focus on dyslipidemia drug targets traceable to human genetic studies, including statins and ezetimibe, as well as promising new classes such as inhibitors of proprotein convertase subtilisin kexin 9, apolipoprotein B, microsomal triglyceride transfer protein, cholesteryl ester transfer protein, angiopoietin like proteins types 3 and 4 and apolipoprotein C-III. Several of these new agents have
Brief summary
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attained or are closing in on "prime-time readiness" for clinical use in specific situations.
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Understanding human genetics is accelerating the search for new drug targets for dyslipidemia and atherosclerosis prevention. DNA sequencing, genome-wide analyses and Mendelian randomization can
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illuminate key regulators in lipid physiology. We review established and emerging dyslipidemia drug targets and their roots in human genetic studies, including statins and ezetimibe, as well as inhibitors of PCSK9, apolipoprotein B, microsomal triglyceride transfer protein, apolipoprotein C-III and several other that are reaching “prime-time readiness” for clinical use.
ACCEPTED MANUSCRIPT 3 Historically, drug discovery has followed a labour-intensive paradigm of biochemically screening a wide range of proprietary compounds. In one version of this paradigm, an agent yielding a positive signal in an in vitro system is further evaluated in pre-clinical in vitro and in vivo models, ascending up
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the phylogenetic tree until finally reaching early phase human trials. However, this is often a frustrating and protracted process as numerous compounds identified in this manner have failed to show
anticipated clinical effects, or have even demonstrated unanticipated off-target toxicities (1). Therefore,
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pharmaceutical research has sought alternative approaches that might better predict success before investing too much time or effort in a particular target or agent. A paradigm shift in the approach to
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drug discovery began about 10 years ago, when the field of human genetics offered pharmaceutical researchers the potential to better predict the efficacy and toxicity of agents that targeted specific pathways or molecules (1). This approach has been fruitful in the area of cardiovascular disease (CVD) and its risk factors, specifically dyslipidemia (2). Studies of common genetic variants (3) and especially of
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rare genetic variants (4) have yielded several leads for drug targets that are under development. Drugs traceable to research in rare familial disorders are shown in Table 1, while targets for lipid and CVD risk
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reduction characterized in genetic epidemiology and population studies are shown in Table 2.
Case study of human genetics informing drug design: statins
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Long before the rise of modern genomics, rare familial disorders provided key insights into lipid metabolism and pathophysiology, allowing for a top-down approach to drug discovery. For instance, the humble statin originated in part from the study of patients with familial hypercholesterolemia (FH), a relatively common genetic disorder characterized by markedly elevated LDL cholesterol and premature cardiovascular mortality (5). The culprit gene in FH was found to encode a defective LDL receptor, impairing hepatic uptake of LDL particles, resulting in their ectopic accumulation within arterial wall macrophages. Once the intracellular pathways were explained, it became clear that blocking production
ACCEPTED MANUSCRIPT 4 of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase would increase hepatic clearance of circulating LDL particles. A structured search for HMGCoA reductase inhibitors yielded compactin (6), which after slight
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chemical modification became lovastatin, the first statin approved for clinical use. Understanding the metabolic pathways in cholesterol metabolism indicated that giving statins to patients with
heterozygous FH would inhibit cholesterol synthesis (7). Since affected individuals had one normally
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functioning LDL receptor allele, it was predicted that HMGCoA reductase inhibition would upregulate residual receptor activity, thus lowering circulating LDL cholesterol. This expectation was borne out in
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early phase clinical trials. Subsequently, the use of statins spread diffusely into the general cardiology patient population - who each have two functional LDL receptor alleles - where a mountain of clinical trial evidence showed reduction both of cardiovascular risk and overall mortality. Statins now occupy a
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central role in guidelines for cardiovascular risk reduction (8).
PCSK9 inhibitors: combining studies of rare families with epidemiologic populations The growing power and depth of modern genetic analysis has helped uncover the basis of
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clinically favorable naturally-occurring human genetic variants. Industry has sought to pharmacologically replicate these “experiments of nature”. Perhaps the most compelling case in point is the development
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of proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors. In 2003, the PSCK9 gene was implicated as causative in families with a rare form of severe hypercholesterolemia resembling FH, due to heterozygous hyper-functional or "gain-of-function" variants (9). Seidah and Chrétien in Montréal had discovered the PCSK9 protease, which normally degrades LDL receptors (10). The rare exuberant gainof-function mutations in PCSK9 were shown to particularly impair the uptake of LDL particles, leading to elevated LDL cholesterol levels (11). Methodical follow-up experiments discovered mirror image "lossof function" variants in this gene, seen in up to 3% of the general population; carriers of these sluggish
ACCEPTED MANUSCRIPT 5 forms of PSCK9 had naturally depressed LDL cholesterol levels and virtually absent atherosclerotic CVD with no apparent adverse effects (12). The random allocation of such favorable "mutations" at the time of meiosis resembled the
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structure of a randomized clinical trial: this has led to the term "Mendelian randomization", in which the effects of a natural genetic "intervention" are studied over a lifetime in an epidemiologic cohort.
Because carriers of the favorable PSCK9 alleles were so clearly protected from atherosclerosis, big
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pharma immediately turned its attention towards developing an intervention to simulate the beneficial effect of the rare genetic variant. PCSK9 was an ideal drug target for inhibition or knockdown. However,
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small molecules (i.e. pills or tablets) do not work on this pathway; an alternate means of delivering an inhibitor was required (13).
In 2010, the first PCSK9 inhibitor trials were undertaken, using parenterally administered monoclonal antibodies (14). Early phase trials of evolocumab, alirocumab, and bococizumab
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demonstrated both short-term safety and unprecedented levels of LDL cholesterol reduction (>70%) (14). Short term outcome studies (15; 16) showed reductions in major CVD events of >50%; these results explain why Health Canada approved evolocumab in 2015 and alirocumab in 2016 for LDL cholesterol
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reduction in patients with FH or in those with very high cardiovascular risk who were not responsive to statins. Large-scale phase 3 trials are underway, with data on clinical outcomes anticipated soon (13),
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although development of bococizumab was recently terminated because of safety concerns. If such LDL cholesterol reductions translate into the expected reductions in cardiovascular
outcomes in large scale prospective studies, PCSK9 therapy could alter the landscape of dyslipidemia treatment and CVD prevention, especially for those with genetic dyslipidemia, or with statin intolerance, or with inadequate response to current therapies. Moreover, newer delivery systems under development could reduce the frequency of administration to once-yearly. At any rate, the progress
ACCEPTED MANUSCRIPT 6 from gene mapping to informative drug trials for PCSK9 inhibitors took less than a decade, highlighting the potential efficiency of genetics-driven drug development.
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Ezetimibe: a drug whose efficacy was retroactively justified by Mendelian randomization
Since it was approved for use in Canada in 2003, ezetimibe has had a somewhat checkered history, since many clinicians felt circumspect about its efficacy in reducing CVD end points (17). These
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reservations were alleviated recently by evidence of the CVD benefits of ezetimibe. Specifically, two clinical trials showed reductions of CVD risk when ezetimibe was used in combination with a statin (18;
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19), the latter of which definitively showed that incremental CVD risk reduction attributable to ezetimibe was directly proportional to its modest LDL cholesterol lowering ability. Concurrently, a large meta-analysis of genetic epidemiologic data applied the Mendelian randomization approach to model the impact of ezetimibe at the genetic level (20). Using next
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generation sequencing technology, the investigators identified some rare individuals who carried DNA variants in the NPC1L1 gene encoding the target for ezetimibe, namely Niemann-Pick C1-like protein 1. Carriers of such inactivating or loss-of-function variants had lifelong lower levels of LDL cholesterol by
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0.31 mmol/L, together with a 53% reduction in myocardial infarction risk (20). It was as if the carriers of these gene variants were born taking ezetimibe: the impact over their lifetime was reduced CVD risk.
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Together with the clinical trial results, these genetic findings demonstrated that LDL cholesterol reduction via a non-statin mechanism can reduce CVD risk (21), which has elevated ezetimibe to a priority position above other non-statin agents in current dyslipidemia guidelines (8).
Abetalipoproteinemia: an ultra-rare condition that led to a potent cholesterol-lowering drug Abetalipoproteinemia (ABL) is a very rare autosomal recessive disorder caused by a mutation in microsomal triglyceride transfer protein (MTP), which is encoded by the MTTP gene (22). In ABL, the
ACCEPTED MANUSCRIPT 7 complete deficiency of the large subunit of MTP results in the inability of the liver and intestine to produce and secrete apolipoprotein (apo B)-containing lipoproteins. LDL and very low density lipoprotein (VLDL) are virtually absent in homozygotes, associated with malabsorption, failure to thrive,
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and systemic sequelae due to fat-soluble vitamin deficiency; however, patients were completely free of atherosclerosis (22). Although complete deficiency of MTP is deleterious, there was reason to believe that partial inhibition may capture the biochemical benefit of reduced LDL cholesterol without the
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systemic adverse effects.
The search began in the early 1990's for a chemical to partially inhibit MTP in those with severe
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FH – just enough to impair the production of LDL, but without wiping it out completely. In 1997, lomitapide was synthesized with precisely these properties, and was developed to a certain point by Bristol Myers Squibb. However, the company halted development when, despite their excellent lipid profile, fatty liver was observed in a large proportion of treated individuals. In retrospect, this is an
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expected complication of the agent based on its mechanism of action: the assembly of lipoprotein precursors is thwarted by inhibition of MTP, and the lipids have to go somewhere. The license for lomitapide was acquired by Aegerion pharmaceuticals in the mid-2000's with the
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idea that the drug might find a niche in patients whose CVD risk was so tremendously high and acute, that it would offset the potential risk of fatty liver. Rare patients with homozygous FH (HoFH) - whose
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untreated LDL cholesterol levels exceeded 10 mmol/L and required biweekly lipoprotein apheresis to be controlled - represented such a patient group (23). It is estimated that there are fewer than 100 HoFH patients in Canada (24). Statins and indeed almost all lipid lowering treatments are minimally effective in these patients; without regular apheresis they succumb to CVD by the second or third decade of life. Subsequent phase 2 (25) and phase 3 trials (26) of lomitapide in patients with HoFH showed potent LDL cholesterol - up to 50%. Gastrointestinal symptoms were common however, and hepatic steatosis was noted in most patients. More severe liver disease was associated with very long term use
ACCEPTED MANUSCRIPT 8 in anecdotal reports (27). However, the risk-benefit ratio in the situation of HoFH was judged to favour the use of lomitapide to reduce virtually certain CVD risk while accepting the less clear longer term risks of transaminase elevation and fatty liver. Because of the minute size of this patient population, a
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prospective, randomized double-blind study of CVD outcomes with lomitapide is unlikely to ever be performed. This daily oral medication was approved by the FDA in 2012 and by Health Canada in 2014 under the trade name Juxtapid (Aegerion) for patients with HoFH. It is possible that more specific
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targeting of the subdomains of MTP may result in next-generation versions of this drug that maximize its biochemical benefits, while minimizing or eliminating its adverse effects (28). Due to its high cost and
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hepatotoxicity, lomitapide is currently reserved for only those with HoFH.
Hypobetalipoproteinemia: another rare LDL cholesterol deficiency state that drove drug development A related condition to ABL is familial hypobetalipoproteinemia (FHBL), which is encountered in
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both heterozygous and homozygous forms (22). In technical terms FHBL is a co-dominant disorder, with each dose of the dysfunctional allele resulting in a progressively more extreme phenotype. While the homozygous form is indistinguishable from ABL, heterozygous FHBL is associated with a very favorable
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lipid profile with apparent longevity, freedom from coronary artery disease, and no obvious deleterious symptoms except possibly hepatic steatosis (29). The causative gene is APOB, which is renowned for
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encoding the protein component of LDL and related particles; indeed apo B levels are an alternative target measurement in dyslipidemia guidelines (8). FHBL patients show that if apo B synthesis is impaired, the precursors of LDL particles cannot be assembled and so production of LDL is aborted at the outset, resulting in lower levels over a patient's lifetime. Because of the favorable lipid and cardiovascular phenotype of individuals with heterozygous FHBL, the pharmaceutical industry identified the APOB gene as a therapeutic target. In this instance, the emerging field of antisense therapy translated the genetic defect in heterozygous FHBL into
ACCEPTED MANUSCRIPT 9 mipomersen, an antisense oligonucleotide (ASO) which intervenes at the mRNA level to inhibit LDL synthesis (30). Mipomersen’s nucleotide sequence hybridizes to the complementary segment of apo B mRNA, promoting its degradation by exonucleases, and thereby preventing translation into apo B.
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Mipomersen is administered by weekly subcutaneous injections. Several trials confirmed mipomersen’s efficacy in lowering LDL cholesterol in adults with dyslipidemia, including those with HoFH (31).
In a recent meta-analysis of eight RCTs, mipomersen was shown to reduce LDL cholesterol, total
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cholesterol and triglycerides by 32%, 24%, 36% respectively (32). It also reinforced a few caveats to its use: hepatic steatosis, injection site reactions and flu-like symptoms were the most common side effects.
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Mipomersen was approved in the US in 2012 for treatment of HoFH, with trade name Kynamro (Genzyme), but was never approved in Canada, possibly related to concerns over its adverse effects. Although mipomersen itself may not pan out to be a widely used agent, it does establish the potential efficacy of anti-sense RNA-based strategies as an alternative platform to monoclonal antibodies as a
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means to specifically target individual molecules in key metabolic pathways (33).
Cholesteryl ester transfer protein: controversy both in its genetics and clinical trials
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Cholesteryl ester transfer protein (CETP) plays a key role in shuttling lipids between circulating lipoprotein particles (34-35). Early genetic studies of families with CETP deficiency resulting from rare
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loss-of-function mutations in the CETP gene showed that patients had markedly elevated levels of highdensity lipoprotein (HDL) cholesterol together with apparent protection from CVD risk (36), while subsequent studies showed that carriers of these mutations had increased CVD risk despite elevated HDL cholesterol levels (37). More recently, Mendelian randomization studies in some populations suggested that carriers of common DNA variants in the CETP gene had increased HDL cholesterol and significantly decreased CVD risk (38), while other similar studies showed either a marginal increase in CVD risk despite favorable HDL cholesterol levels (39) or no change in CVD risk (40).
ACCEPTED MANUSCRIPT 10 The contradictory genetic findings have been paralleled by inconsistent clinical trial experience with small molecule inhibitors of CETP. Torcetrapib raised HDL cholesterol levels, but also increased risk of CVD events and deaths (41), possibly related to off-target adverse effects. Dalcetrapib showed less
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potent HDL cholesterol raising efficacy but was neutral with respect to CVD outcomes (42). Apparently similar neutral results were seen with evacetrapib despite an even greater mean HDL cholesterol
increase (43). While the aggregate of results raise doubts and questions about the HDL hypothesis (44),
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the lipid community awaits the report of CVD outcomes in an RCT of the last standing CETP inhibitor,
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namely anacetrapib, which also reduces LDL cholesterol; these are anticipated in the next year (45).
Familial combined hypolipidemia and evinacumab
Familial combined hypolipidemia provided another promising target for biologic therapy, leading to the development of evinacumab. Familial combined hypolipidemia is a relatively recently
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recognized autosomal recessive disorder characterized by globally reduced levels of LDL and HDL cholesterol and triglyceride, with no apparent adverse effects. The gene was found by screening families that appeared to have heterozygous FHBL, but without a mutation in the APOB gene. The hypolipidemia
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phenotype is caused by rare loss-of-function mutations affecting the ANGPTL3 gene encoding angiopoietin like protein 3 (46). ANGPTL3 is a glycoprotein that reversibly inhibits lipoprotein lipase (LPL),
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which is responsible for the hydrolysis of triglycerides within lipoproteins and clearance of chylomicrons and VLDL from blood; it also inhibits endothelial lipase, which hydrolyzes HDL (47). These mechanisms are consistent with the previous findings that gain-of-function experiments with ANGPTL3 increased total cholesterol and triglyceride levels in mice, while loss-of-function mutations were found in those in the lowest quartile of triglycerides in the Dallas Heart Study (48). Evinacumab (REGN1500, Regeneron), a human monoclonal antibody that inhibits ANGPTL3 is currently undergoing a phase 2 trial in patients with HoFH. Preliminary results reported in June 2016 showed dramatic LDL cholesterol reductions when
ACCEPTED MANUSCRIPT 11 evinacumab was added to existing therapy in three HoFH patients enrolled, with no serious adverse events identified except for injection site reactions (49). There is also some reason to believe the agent
development.
Apolipoprotein C-III deficiency and volanesorsen
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would be effective in severe hypertriglyceridemia. Anti-sense therapies against ANGPTL3 are also in
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The apparently successful application of the strategy to identify drug targets based on favorable clinical phenotypes in carriers of inactivated gene products is further exemplified by apo C-III deficiency.
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The APOC3 gene has been known to be a key determinant of plasma lipids for > 30 years (50). A study in a Pennsylvania Amish population found that carriers of a rare APOC3 gene knock-out variant had favorable lipids and decreased atherosclerosis (51). These findings were extended in two epidemiologic studies using DNA sequencing in populations under a Mendelian randomization framework, which both
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found that carriers of rare DNA variants encoding inactivated apo C-III had improved lipids and decreased lifetime CVD risk (52-53). Both monoclonal antibodies and anti-sense RNA strategies were rapidly pursued in an effort to simulate the effects of genetically knocked down apo C-III. The agent that
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is furthest along the development pathway is the anti-sense RNA drug volanesorsen (Ionis/Akcea), which in two reported early phase trials has been shown to have a potent effect on reducing plasma lipids,
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particularly severely elevated triglyceride levels (54-55). Concerns include injection site reactions and effects on hematologic variables. Whether such treatment could be applied more widely to reduce CVD risk in at-risk patients with mildly to moderately elevated lipids, already treated on a statin will certainly require definitive outcomes studies.
Human gene therapy for severe dyslipidemia
ACCEPTED MANUSCRIPT 12 Most examples above illustrate the approach of finding human deficiency states that are associated with favorable clinical phenotypes and then developing pharmacologic agents to mimic these beneficial genetic effects. The reasoning behind this approach is that typically it is easier to
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pharmacologically inhibit or disrupt a target molecule, rather than selectively boost or stimulate its activity. With targeted biologics, such as monoclonal antibodies or antisense strategies, it becomes even more feasible to precisely target the specific gene or gene product that is indicated to be clinically
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relevant by the human genetic deficiency state.
However, there are at least two examples where a human deficiency that results in a clinically
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deleterious phenotype can be corrected at least somewhat by replacing the deficient gene, although space does not permit comprehensive discussion here. First, HoFH, as mentioned above, results from virtually complete deficiency of LDL receptor activity. Promising studies of adeno-associated virus-based gene therapy in this condition in primate models (56) have resulted in accelerated development of this
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type of investigational treatment in humans (AAV8.TBG.hLDLR; RegenXbio), to be administered by intravenous infusion. Second, familial lipoprotein lipase (LPL) deficiency is an ultra-rare disorder (probably < 100 Canadian patients) due to bi-allelic mutations that abolish enzyme function and result in
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profound hypertriglyceridemia with high risk of death from pancreatitis (57). Alipogene tiparvovec (Glybera, uniQure BV) is an adeno-associated virus gene therapy that was developed to treat patients
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with LPL deficiency. It is administered intramuscularly and has recently shown some evidence of long term benefit (58). Glybera has been approved since 2012 in Europe for treatment of this condition, but has not yet been approved in North America.
Enzyme replacement in lysosomal acid lipase deficiency Lysosomal acid lipase deficiency (LALD) is an ultra-rare disorder (probably < 100 Canadian patients) due to bi-allelic mutations in the LIPA gene encoding lysosomal acid lipase (also known as
ACCEPTED MANUSCRIPT 13 cholesterol ester hydrolase). The mutations abolish function of the enzyme, resulting in combined dyslipidemia and liver injury due to cholesterol ester accumulation (59). While gene therapy is one theoretical approach to replace the missing enzyme, in this condition, the actual enzyme itself was
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developed as a drug (sebelipase alfa, Synageva) to be given by intermittent intravenous infusions. A recent phase 3 clinical trial conducted in 66 patients with LALD showed significant improvement in transaminases and reduction in hepatic lipid content (60). Kanuma (Alexion) was approved in 2015 by
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the US Food and Drug Administration and the European Medicines Agency for treatment of LALD, but has not yet been approved in Canada. Cost is an important consideration, as it is with all orphan drugs
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for ultra-rare conditions.
Angiopoietin like protein 4: a drug target candidate recently nominated by genetics Inactivating DNA variants in the ANGPTL4 gene that encodes angiopoietin like protein 4 were
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recently reported to be associated with a favorable lipid profile, including reduced triglycerides and raised HDL cholesterol together with reduced CVD risk in epidemiological samples (61-62) and also in patients ascertained through electronic health records (63). However, before jumping on the
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bandwagon to develop agents to inhibit ANGPTL4, it is worth recalling that early animal studies with a monoclonal antibody to ANGPTL4 were associated with development of intestinal lipomatosis and
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granulomatosis (64), which were also observed more recently when mice and monkeys were treated with a similar antibody (63). This example illustrates how knowledge from other non-genetic biological data needs to be integrated into the decision-making process when gauging the potential value of drug targets identified using purely genetic approaches (48).
Apolipoprotein A-V: implicated by genetics
ACCEPTED MANUSCRIPT 14 Apo A-V encoded by the APOA5 gene modulates the metabolism of triglyceride rich lipoproteins. A gargantuan DNA sequencing study found that carriers of rare inactivating APOA5 variants had ~2.2fold increased risk of early myocardial infarction, explaining ~1-2% of total cases (65). APOA5 thus
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represents another potential drug target (66), but the challenge here is that the favorable clinical
phenotype requires increased expression of the protein. A new drug would thus need to selectively boost or stimulate apo A-V production, which is more challenging to accomplish than knocking it down.
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In addition, except in ultra-rare cases of complete deficiency of apo A-V associated with severe
hypertriglyceridemia (58), most "garden variety" cases from the Mendelian randomization studies are
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heterozygous (65), with presumably only relative deficiency of apo A-V. Furthermore, plasma levels of apo A-V cover a very wide range in the "healthy" general population (66), so the therapeutic goal or target level from upregulating the gene product is uncertain at best.
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Identification of other lipid-based drug targets
We have described several examples where human genetics has pointed the way - or is pointing the way - to potentially useful new therapeutics focused on lipids that may eventually have a beneficial
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impact on hard clinical outcomes. These were largely based on rare monogenic variants (67). However, in addition to these targets, other types of genetic studies (3) have identified a plethora of additional
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variants, of which at least some might be potential targets for further consideration and development. For instance, genome-wide association studies (GWAS) of common genetic variants determining
plasma lipid levels in the general population have identified 157 highly significant gene loci out of millions that were tested (68-69). Many of these significant loci are already familiar determinants, including LDLR, HMGCR, APOB, PSCK9, ANGPTL3, LPL, APOA5, LPA and CETP, but more than half of them are within gene loci that are poorly characterized. An optimistic view is that some of the novel "hits"
ACCEPTED MANUSCRIPT 15 from GWAS will turn out to be clinically relevant. A harbinger of this relevance is that lipid-related loci represent ~ 25% of all significantly associated genes from GWAS of coronary artery disease (70).
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Conclusions
After more than a decade characterized by few new developments, and mainly by genericization of statins, the past 18 months has witnessed a re-energization of the lipid field, with approval of PCSK9
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inhibitors and some other specialty drugs for management of dyslipidemia in particular contexts.
Whether the PCSK9 inhibitors ever become more widely used will depend on results of upcoming end
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point studies. But perhaps equally striking is that there is now a rich development pipeline for even newer agents that target lipid-related genes and gene products, which originally came to attention through the study of human genetics (71). Some of these newer agents may only have niche or orphan indications. But the same could have been said of statins 30 years ago, when their initial use was
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restricted to lipid and metabolic clinics, but through medical progress and mounting clinical trial evidence, spread to the general cardiology population. At least four lipid-lowering agents mentioned in the 2016 Canadian Lipid Guidelines - statins, ezetimibe, PSCK9 inhibitors, and lomitapide (72) - have
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development stories that are traceable to human genetics, with perhaps more examples pending.
ACCEPTED MANUSCRIPT 16 Acknowledgements/disclosures RAH is supported by the Jacob J. Wolfe Distinguished Medical Research Chair, the Edith Schulich Vinet
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Research Chair in Human Genetics, and the Martha G. Blackburn Chair in Cardiovascular Research.
RAH has received operating grants from the Canadian Institutes of Health Research (Foundation Grant), the Heart and Stroke Foundation of Ontario (T-000353), and Genome Canada through Genome Quebec
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(award 4530).
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RAH has received honoraria for membership on advisory boards and speakers' bureaus for Aegerion, Amgen, Gemphire, Lilly, Merck, Pfizer, Regeneron, Sanofi and Valeant.
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LRW has no disclosures to report.
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ACCEPTED MANUSCRIPT 19 27. Sacks FM, Stanesa M, Hegele RA. Severe hypertriglyceridemia with pancreatitis: thirteen years' treatment with lomitapide. JAMA Intern Med 2014; 174:443-7. 28. Burnett JR, Hegele RA. Finding the Therapeutic Sweet Spot: Using Naturally Occurring Human Variants to Inform Drug Design. Circ Cardiovasc Genet 2015; 8:637-9.
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39. Voight BF, Peloso GM, Orho-Melander M, et al. Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomisation study. Lancet 2012; 380:572-80. 40. Wu Z, Lou Y, Qiu X, et al. Association of cholesteryl ester transfer protein (CETP) gene polymorphism, high density lipoprotein cholesterol and risk of coronary artery disease: a meta-analysis using a Mendelian randomization approach. BMC Med Genet 2014; 15:118.
ACCEPTED MANUSCRIPT 20 41. Barter PJ, Caulfield M, Eriksson M, et al. Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med 2007; 357:2109-22. 42. Schwartz GG, Olsson AG, Abt M, et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med 2012; 367:2089-99.
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43. Nicholls SJ, Lincoff AM, Barter PJ, et al. Assessment of the clinical effects of cholesteryl ester transfer protein inhibition with evacetrapib in patients at high-risk for vascular outcomes: Rationale and design of the ACCELERATE trial. Am Heart J 2015; 170:1061-9.
44. Ng DS, Wong NC, Hegele RA. HDL - is it too big to fail? Nat Rev Endocrinol 2013 ;9:308-12.
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45. Barter PJ, Rye KA. Cholesteryl Ester Transfer Protein Inhibition Is Not Yet Dead--Pro. Arterioscler Thromb Vasc Biol 2016; 36:439-41.
46. Pisciotta L, Favari E, Magnolo L, et al. Characterization of three kindreds with familial combined
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hypolipidemia caused by loss-of-function mutations of ANGPTL3. Circ Cardiovasc Genet 2012; 5:42-50. 47. Hegele RA. Multidimensional regulation of lipoprotein lipase: impact on biochemical and cardiovascular phenotypes. J Lipid Res 2016; 57:1601-7.
48. Victor RG, Haley RW, Willett DL, et al. The Dallas Heart Study: a population-based probability sample for the multidisciplinary study of ethnic differences in cardiovascular health. Am J Cardiol 2004; 93:147380.
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49. Gaudet D, Gipe DA, Khoury É et al. Safety and efficacy of evinacumab, a monoclonal antibody to ANGPTL3, in patients with homozygous familial hypercholesterolemia: a single arm, open-label, proof of concept study. 84th Congress, European Atherosclerosis Society, Innsbruck Austria, 1 June 2016, abstract, oral presentation.
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50. Huff MW, Hegele RA. Apolipoprotein C-III: going back to the future for a lipid drug target. Circ Res
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51. Pollin TI, Damcott CM, Shen H, et al. A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 2008; 322:1702-5. 52. Jørgensen AB, Frikke-Schmidt R, Nordestgaard BG, Tybjærg-Hansen A. Loss-of-function mutations in APOC3 and risk of ischemic vascular disease N Engl J Med. 2014; 371:32-41. 53. Crosby J, Peloso GM, Auer PL, et al. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med 2014; 371:22-31. 54. Gaudet D, Brisson D, Tremblay K, et al. Targeting APOC3 in the familial chylomicronemia syndrome. N Engl J Med 2014; 371:2200-6.
ACCEPTED MANUSCRIPT 21 55. Gaudet D, Alexander VJ, Baker BF, et al. Antisense Inhibition of apolipoprotein C-III in patients with hypertriglyceridemia. N Engl J Med 2015; 373:438-47. 56. Somanathan S, Jacobs F, Wang Q, Hanlon AL, Wilson JM, Rader DJ. AAV vectors expressing LDLR gain-of-function variants demonstrate increased efficacy in mouse models of familial
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hypercholesterolemia. Circ Res 2014; 115:591-9.
57. Brahm AJ, Hegele RA. Chylomicronaemia--current diagnosis and future therapies. Nat Rev Endocrinol 2015; 11:352-62.
58. Gryn SE, Hegele RA. Novel therapeutics in hypertriglyceridemia. Curr Opin Lipidol 2015; 26:484-91.
spectrum. Mol Genet Metab 2008; 93:282-94.
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59. Rahalkar AR, Hegele RA. Monogenic pediatric dyslipidemias: classification, genetics and clinical
deficiency. N Engl J Med 2015; 373:1010-20.
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60. Burton BK, Balwani M, Feillet F, et al. A phase 3 trial of sebelipase alfa in lysosomal acid lipase
61. Stitziel NO, Stirrups KE, Masca NG, et al. Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease. N Engl J Med 2016; 374:1134-44.
62. Helgadottir A, Gretarsdottir S, Thorleifsson G, et al. Variants with large effects on blood lipids and the role of cholesterol and triglycerides in coronary disease. Nat Genet 2016; 48:634-9. 63. Dewey FE, Gusarova V, O'Dushlaine C, et al. Inactivating variants in ANGPTL4 and risk of coronary
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artery disease. N Engl J Med 2016; 374:1123-33.
64. Desai U, Lee EC, Chung K, et al. Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice. Proc Natl Acad Sci USA 2007; 104:11766-71.
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65. Do R, Stitziel NO, Won HH, et al. Exome sequencing identifies rare LDLR and APOA5 alleles conferring risk for myocardial infarction. Nature 2015; 518:102-6.
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66. Forte TM, Sharma V, Ryan RO. Apolipoprotein A-V gene therapy for disease prevention / treatment: a critical analysis. J Biomed Res 2015; 30 doi: 10.7555/JBR.30.20150059 67. Hegele RA, Ban MR, Cao H, McIntyre AD, Robinson JF, Wang J. Targeted next-generation sequencing in monogenic dyslipidemias. Curr Opin Lipidol 2015; 26:103-13. 68. Teslovich TM, Musunuru K, Smith AV, et al. Biological, clinical and population relevance of 95 loci for blood lipids. Nature 2010; 466:707-13. 69. Willer CJ, Schmidt EM, Sengupta S, et al. Discovery and refinement of loci associated with lipid levels. Nat Genet 2013; 45:1274-83. 70. McPherson R, Tybjaerg-Hansen A. Genetics of coronary artery disease. Circ Res 2016; 118:564-78.
ACCEPTED MANUSCRIPT 22 71. Kathiresan S. Developing medicines that mimic the natural successes of the human genome: lessons from NPC1L1, HMGCR, PCSK9, APOC3, and CETP. J Am Coll Cardiol 2015; 65:1562-6. 72. Anderson TJ, Grégoire J, Pearson GJ, et al. 2016 Canadian Cardiovascular Society Guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult. Can J Cardiol
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23
MTTP
1990
Hypobetalipoproteinemia
APOB
1983
Proprotein convertase subtilisin kexin 9 (PSCK9) deficiency
PCSK9
2003
Cholesteryl ester transfer protein (CETP) deficiency
CETP
1989
Familial combined hypolipidemia
ANGPTL3
2001
Apolipoprotein C-III deficiency
APOC3
Lipoprotein lipase deficiency
LPL
Elevated LDL cholesterol Depressed apo Bcontaining lipoproteins Depressed apo Bcontaining lipoproteins Low LDL cholesterol
Elevated HDL cholesterol; low LDL cholesterol Low LDL cholesterol and triglycerides Low triglycerides and elevated HDL cholesterol Elevated triglycerides
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1985
Stage of development Approved worldwide between 1987 and 2001 (pitavastatin not approved in Canada). Now all are genericized in Canada. They are the standard of care in CVD prevention.
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1981
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LDLR
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Familial hypercholesterolemia Abetalipoproteinemia
Resulting therapeutic(s) statins (lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin, pitavastatin) LDL receptor gene therapy lomitapide (Juxtapid)
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Table 1. Lipid therapies that can be traced to study of familial disorders Rare familial disorder Implicated Approximate year Biochemical gene name of gene cloning* phenotype Familial LDLR 1981 Elevated LDL hypercholesterolemia cholesterol
mipomersen (Kynamro)
Phase 2 clinical trials underway in 2016. Approved in the US and Europe in 2013, and in Canada in 2014 for homozygous familial hypercholesterolemia (Juxtapid). Approved in US only in 2013 for homozygous familial hypercholesterolemia (Kynamro).
evolocumab (Repatha), alirocumab (Praluent), bococizumab anacetrapib
Approval worldwide as of 2015. In Canada evolocumab (Repatha) approved in 2015 and alirocumab (Praluent) approved in 2016. Development of bococizumab terminated in November 2016 due to safety concerns. Not yet approved. Phase 3 clinical trials since 2003.
evinacumab
Not yet approved. Phase 2/3 clinical trials since 2015.
volanesorsen
Not yet approved. Phase 2/3 clinical trials since 2013.
alipogene Approved in Europe in 2012 (Glybera). tiparvovec (Glybera) * or linkage of the gene to the human disease phenotype; abbreviations: LDL, low density lipoprotein; apo, apolipoprotein; CVD, cardiovascular disease
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24 Table 2. Examples of lipid drug targets that have been validated in Mendelian randomization studies Lipid effect
HMGCR
Loss-of-function: lowers LDL cholesterol (also increases weight and diabetes risk)
PSCK9
Loss-of-function: lowers LDL cholesterol
Loss-of-function: lowers CHD risk
NPC1L1
Loss-of-function: lowers LDL cholesterol
Loss-of-function: lowers CHD risk
APOC3
Loss-of-function: lowers TG, raises HDL cholesterol Loss-of-function: lowers TG, raises HDL cholesterol
Loss-of-function: lowers CHD risk Loss-of-function: lowers CHD risk
Loss-of-function: raises TG, lowers HDL cholesterol Gain-of-function: raises Lp(a) levels
Loss-of-function: increases CHD risk Gain-of-function: increases CHD risk
LPA
Inhibitors lower both LDL cholesterol and CHD risk in virtually all statin trials in both primary and secondary prevention. Inhibitors lowers both LDL cholesterol and CHD risk in metaanalysis of phase 3 studies. Inhibitor lowers both LDL cholesterol and CHD risk on top of statin (e.g. ezetimibe in the IMPROVE-IT study). Inhibitor lowers TG levels; CHD study not done yet. Inhibitor improves lipid profile, but may have off-target effects; CHD study not done yet. Not studied yet.
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APOA5
CHD effect in clinical trials
Inhibitor reduces Lp(a) levels selectively, CHD study not done yet.
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ANGPTL4
CHD effect in genetic studies Loss-of-function: lowers CHD risk
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Target
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abbreviations: CHD, coronary heart disease; LDL, lower density lipoprotein; TG, triglyceride
S0828-282X(16)31088-1
DOI:
10.1016/j.cjca.2016.11.003
Reference:
CJCA 2300
To appear in:
Canadian Journal of Cardiology
Received Date: 19 September 2016 Revised Date:
6 November 2016
Accepted Date: 7 November 2016
Please cite this article as: Wang LR, Hegele RA, Genetics for the identification of lipid targets - beyond PCSK9, Canadian Journal of Cardiology (2016), doi: 10.1016/j.cjca.2016.11.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Department of Medicine and Robarts Research Institute, Schulich School of Medicine and Dentistry,
Correspondence: Robert Hegele MD, FRCPC, FACP, FCCS Robarts Research Institute
London, Ontario N6A 5B7
tel: 519-931-5271
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fax: 519-931-5218
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email: [email protected]
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4288A - 1151 Richmond Street North
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Western University, London, Ontario N6A 5B7
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Linda R. Wang MD and Robert A. Hegele MD, FRCPC
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Genetics for the identification of lipid targets - beyond PCSK9
ACCEPTED MANUSCRIPT 2 Abstract From studies of rare families to genome-wide associations in populations, understanding of human genetics has accelerated the search for new drug targets for the prevention of atherosclerotic
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cardiovascular disease. DNA sequencing and genome-wide analyses of DNA markers have illuminated both rare and common variants in genes that regulate lipids and ultimately atherosclerosis risk. A
recent innovative approach called Mendelian randomization can endorse specific genes and variants as
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causative not just for lipid disturbances, but also for clinical cardiovascular end points. This knowledge helps prioritize the candidate genes and proteins in the drug development pipeline. In this review, we
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focus on dyslipidemia drug targets traceable to human genetic studies, including statins and ezetimibe, as well as promising new classes such as inhibitors of proprotein convertase subtilisin kexin 9, apolipoprotein B, microsomal triglyceride transfer protein, cholesteryl ester transfer protein, angiopoietin like proteins types 3 and 4 and apolipoprotein C-III. Several of these new agents have
Brief summary
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attained or are closing in on "prime-time readiness" for clinical use in specific situations.
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Understanding human genetics is accelerating the search for new drug targets for dyslipidemia and atherosclerosis prevention. DNA sequencing, genome-wide analyses and Mendelian randomization can
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illuminate key regulators in lipid physiology. We review established and emerging dyslipidemia drug targets and their roots in human genetic studies, including statins and ezetimibe, as well as inhibitors of PCSK9, apolipoprotein B, microsomal triglyceride transfer protein, apolipoprotein C-III and several other that are reaching “prime-time readiness” for clinical use.
ACCEPTED MANUSCRIPT 3 Historically, drug discovery has followed a labour-intensive paradigm of biochemically screening a wide range of proprietary compounds. In one version of this paradigm, an agent yielding a positive signal in an in vitro system is further evaluated in pre-clinical in vitro and in vivo models, ascending up
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the phylogenetic tree until finally reaching early phase human trials. However, this is often a frustrating and protracted process as numerous compounds identified in this manner have failed to show
anticipated clinical effects, or have even demonstrated unanticipated off-target toxicities (1). Therefore,
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pharmaceutical research has sought alternative approaches that might better predict success before investing too much time or effort in a particular target or agent. A paradigm shift in the approach to
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drug discovery began about 10 years ago, when the field of human genetics offered pharmaceutical researchers the potential to better predict the efficacy and toxicity of agents that targeted specific pathways or molecules (1). This approach has been fruitful in the area of cardiovascular disease (CVD) and its risk factors, specifically dyslipidemia (2). Studies of common genetic variants (3) and especially of
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rare genetic variants (4) have yielded several leads for drug targets that are under development. Drugs traceable to research in rare familial disorders are shown in Table 1, while targets for lipid and CVD risk
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reduction characterized in genetic epidemiology and population studies are shown in Table 2.
Case study of human genetics informing drug design: statins
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Long before the rise of modern genomics, rare familial disorders provided key insights into lipid metabolism and pathophysiology, allowing for a top-down approach to drug discovery. For instance, the humble statin originated in part from the study of patients with familial hypercholesterolemia (FH), a relatively common genetic disorder characterized by markedly elevated LDL cholesterol and premature cardiovascular mortality (5). The culprit gene in FH was found to encode a defective LDL receptor, impairing hepatic uptake of LDL particles, resulting in their ectopic accumulation within arterial wall macrophages. Once the intracellular pathways were explained, it became clear that blocking production
ACCEPTED MANUSCRIPT 4 of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase would increase hepatic clearance of circulating LDL particles. A structured search for HMGCoA reductase inhibitors yielded compactin (6), which after slight
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chemical modification became lovastatin, the first statin approved for clinical use. Understanding the metabolic pathways in cholesterol metabolism indicated that giving statins to patients with
heterozygous FH would inhibit cholesterol synthesis (7). Since affected individuals had one normally
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functioning LDL receptor allele, it was predicted that HMGCoA reductase inhibition would upregulate residual receptor activity, thus lowering circulating LDL cholesterol. This expectation was borne out in
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early phase clinical trials. Subsequently, the use of statins spread diffusely into the general cardiology patient population - who each have two functional LDL receptor alleles - where a mountain of clinical trial evidence showed reduction both of cardiovascular risk and overall mortality. Statins now occupy a
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central role in guidelines for cardiovascular risk reduction (8).
PCSK9 inhibitors: combining studies of rare families with epidemiologic populations The growing power and depth of modern genetic analysis has helped uncover the basis of
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clinically favorable naturally-occurring human genetic variants. Industry has sought to pharmacologically replicate these “experiments of nature”. Perhaps the most compelling case in point is the development
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of proprotein convertase subtilisin kexin 9 (PCSK9) inhibitors. In 2003, the PSCK9 gene was implicated as causative in families with a rare form of severe hypercholesterolemia resembling FH, due to heterozygous hyper-functional or "gain-of-function" variants (9). Seidah and Chrétien in Montréal had discovered the PCSK9 protease, which normally degrades LDL receptors (10). The rare exuberant gainof-function mutations in PCSK9 were shown to particularly impair the uptake of LDL particles, leading to elevated LDL cholesterol levels (11). Methodical follow-up experiments discovered mirror image "lossof function" variants in this gene, seen in up to 3% of the general population; carriers of these sluggish
ACCEPTED MANUSCRIPT 5 forms of PSCK9 had naturally depressed LDL cholesterol levels and virtually absent atherosclerotic CVD with no apparent adverse effects (12). The random allocation of such favorable "mutations" at the time of meiosis resembled the
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structure of a randomized clinical trial: this has led to the term "Mendelian randomization", in which the effects of a natural genetic "intervention" are studied over a lifetime in an epidemiologic cohort.
Because carriers of the favorable PSCK9 alleles were so clearly protected from atherosclerosis, big
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pharma immediately turned its attention towards developing an intervention to simulate the beneficial effect of the rare genetic variant. PCSK9 was an ideal drug target for inhibition or knockdown. However,
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small molecules (i.e. pills or tablets) do not work on this pathway; an alternate means of delivering an inhibitor was required (13).
In 2010, the first PCSK9 inhibitor trials were undertaken, using parenterally administered monoclonal antibodies (14). Early phase trials of evolocumab, alirocumab, and bococizumab
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demonstrated both short-term safety and unprecedented levels of LDL cholesterol reduction (>70%) (14). Short term outcome studies (15; 16) showed reductions in major CVD events of >50%; these results explain why Health Canada approved evolocumab in 2015 and alirocumab in 2016 for LDL cholesterol
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reduction in patients with FH or in those with very high cardiovascular risk who were not responsive to statins. Large-scale phase 3 trials are underway, with data on clinical outcomes anticipated soon (13),
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although development of bococizumab was recently terminated because of safety concerns. If such LDL cholesterol reductions translate into the expected reductions in cardiovascular
outcomes in large scale prospective studies, PCSK9 therapy could alter the landscape of dyslipidemia treatment and CVD prevention, especially for those with genetic dyslipidemia, or with statin intolerance, or with inadequate response to current therapies. Moreover, newer delivery systems under development could reduce the frequency of administration to once-yearly. At any rate, the progress
ACCEPTED MANUSCRIPT 6 from gene mapping to informative drug trials for PCSK9 inhibitors took less than a decade, highlighting the potential efficiency of genetics-driven drug development.
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Ezetimibe: a drug whose efficacy was retroactively justified by Mendelian randomization
Since it was approved for use in Canada in 2003, ezetimibe has had a somewhat checkered history, since many clinicians felt circumspect about its efficacy in reducing CVD end points (17). These
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reservations were alleviated recently by evidence of the CVD benefits of ezetimibe. Specifically, two clinical trials showed reductions of CVD risk when ezetimibe was used in combination with a statin (18;
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19), the latter of which definitively showed that incremental CVD risk reduction attributable to ezetimibe was directly proportional to its modest LDL cholesterol lowering ability. Concurrently, a large meta-analysis of genetic epidemiologic data applied the Mendelian randomization approach to model the impact of ezetimibe at the genetic level (20). Using next
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generation sequencing technology, the investigators identified some rare individuals who carried DNA variants in the NPC1L1 gene encoding the target for ezetimibe, namely Niemann-Pick C1-like protein 1. Carriers of such inactivating or loss-of-function variants had lifelong lower levels of LDL cholesterol by
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0.31 mmol/L, together with a 53% reduction in myocardial infarction risk (20). It was as if the carriers of these gene variants were born taking ezetimibe: the impact over their lifetime was reduced CVD risk.
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Together with the clinical trial results, these genetic findings demonstrated that LDL cholesterol reduction via a non-statin mechanism can reduce CVD risk (21), which has elevated ezetimibe to a priority position above other non-statin agents in current dyslipidemia guidelines (8).
Abetalipoproteinemia: an ultra-rare condition that led to a potent cholesterol-lowering drug Abetalipoproteinemia (ABL) is a very rare autosomal recessive disorder caused by a mutation in microsomal triglyceride transfer protein (MTP), which is encoded by the MTTP gene (22). In ABL, the
ACCEPTED MANUSCRIPT 7 complete deficiency of the large subunit of MTP results in the inability of the liver and intestine to produce and secrete apolipoprotein (apo B)-containing lipoproteins. LDL and very low density lipoprotein (VLDL) are virtually absent in homozygotes, associated with malabsorption, failure to thrive,
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and systemic sequelae due to fat-soluble vitamin deficiency; however, patients were completely free of atherosclerosis (22). Although complete deficiency of MTP is deleterious, there was reason to believe that partial inhibition may capture the biochemical benefit of reduced LDL cholesterol without the
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systemic adverse effects.
The search began in the early 1990's for a chemical to partially inhibit MTP in those with severe
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FH – just enough to impair the production of LDL, but without wiping it out completely. In 1997, lomitapide was synthesized with precisely these properties, and was developed to a certain point by Bristol Myers Squibb. However, the company halted development when, despite their excellent lipid profile, fatty liver was observed in a large proportion of treated individuals. In retrospect, this is an
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expected complication of the agent based on its mechanism of action: the assembly of lipoprotein precursors is thwarted by inhibition of MTP, and the lipids have to go somewhere. The license for lomitapide was acquired by Aegerion pharmaceuticals in the mid-2000's with the
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idea that the drug might find a niche in patients whose CVD risk was so tremendously high and acute, that it would offset the potential risk of fatty liver. Rare patients with homozygous FH (HoFH) - whose
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untreated LDL cholesterol levels exceeded 10 mmol/L and required biweekly lipoprotein apheresis to be controlled - represented such a patient group (23). It is estimated that there are fewer than 100 HoFH patients in Canada (24). Statins and indeed almost all lipid lowering treatments are minimally effective in these patients; without regular apheresis they succumb to CVD by the second or third decade of life. Subsequent phase 2 (25) and phase 3 trials (26) of lomitapide in patients with HoFH showed potent LDL cholesterol - up to 50%. Gastrointestinal symptoms were common however, and hepatic steatosis was noted in most patients. More severe liver disease was associated with very long term use
ACCEPTED MANUSCRIPT 8 in anecdotal reports (27). However, the risk-benefit ratio in the situation of HoFH was judged to favour the use of lomitapide to reduce virtually certain CVD risk while accepting the less clear longer term risks of transaminase elevation and fatty liver. Because of the minute size of this patient population, a
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prospective, randomized double-blind study of CVD outcomes with lomitapide is unlikely to ever be performed. This daily oral medication was approved by the FDA in 2012 and by Health Canada in 2014 under the trade name Juxtapid (Aegerion) for patients with HoFH. It is possible that more specific
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targeting of the subdomains of MTP may result in next-generation versions of this drug that maximize its biochemical benefits, while minimizing or eliminating its adverse effects (28). Due to its high cost and
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hepatotoxicity, lomitapide is currently reserved for only those with HoFH.
Hypobetalipoproteinemia: another rare LDL cholesterol deficiency state that drove drug development A related condition to ABL is familial hypobetalipoproteinemia (FHBL), which is encountered in
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both heterozygous and homozygous forms (22). In technical terms FHBL is a co-dominant disorder, with each dose of the dysfunctional allele resulting in a progressively more extreme phenotype. While the homozygous form is indistinguishable from ABL, heterozygous FHBL is associated with a very favorable
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lipid profile with apparent longevity, freedom from coronary artery disease, and no obvious deleterious symptoms except possibly hepatic steatosis (29). The causative gene is APOB, which is renowned for
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encoding the protein component of LDL and related particles; indeed apo B levels are an alternative target measurement in dyslipidemia guidelines (8). FHBL patients show that if apo B synthesis is impaired, the precursors of LDL particles cannot be assembled and so production of LDL is aborted at the outset, resulting in lower levels over a patient's lifetime. Because of the favorable lipid and cardiovascular phenotype of individuals with heterozygous FHBL, the pharmaceutical industry identified the APOB gene as a therapeutic target. In this instance, the emerging field of antisense therapy translated the genetic defect in heterozygous FHBL into
ACCEPTED MANUSCRIPT 9 mipomersen, an antisense oligonucleotide (ASO) which intervenes at the mRNA level to inhibit LDL synthesis (30). Mipomersen’s nucleotide sequence hybridizes to the complementary segment of apo B mRNA, promoting its degradation by exonucleases, and thereby preventing translation into apo B.
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Mipomersen is administered by weekly subcutaneous injections. Several trials confirmed mipomersen’s efficacy in lowering LDL cholesterol in adults with dyslipidemia, including those with HoFH (31).
In a recent meta-analysis of eight RCTs, mipomersen was shown to reduce LDL cholesterol, total
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cholesterol and triglycerides by 32%, 24%, 36% respectively (32). It also reinforced a few caveats to its use: hepatic steatosis, injection site reactions and flu-like symptoms were the most common side effects.
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Mipomersen was approved in the US in 2012 for treatment of HoFH, with trade name Kynamro (Genzyme), but was never approved in Canada, possibly related to concerns over its adverse effects. Although mipomersen itself may not pan out to be a widely used agent, it does establish the potential efficacy of anti-sense RNA-based strategies as an alternative platform to monoclonal antibodies as a
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means to specifically target individual molecules in key metabolic pathways (33).
Cholesteryl ester transfer protein: controversy both in its genetics and clinical trials
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Cholesteryl ester transfer protein (CETP) plays a key role in shuttling lipids between circulating lipoprotein particles (34-35). Early genetic studies of families with CETP deficiency resulting from rare
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loss-of-function mutations in the CETP gene showed that patients had markedly elevated levels of highdensity lipoprotein (HDL) cholesterol together with apparent protection from CVD risk (36), while subsequent studies showed that carriers of these mutations had increased CVD risk despite elevated HDL cholesterol levels (37). More recently, Mendelian randomization studies in some populations suggested that carriers of common DNA variants in the CETP gene had increased HDL cholesterol and significantly decreased CVD risk (38), while other similar studies showed either a marginal increase in CVD risk despite favorable HDL cholesterol levels (39) or no change in CVD risk (40).
ACCEPTED MANUSCRIPT 10 The contradictory genetic findings have been paralleled by inconsistent clinical trial experience with small molecule inhibitors of CETP. Torcetrapib raised HDL cholesterol levels, but also increased risk of CVD events and deaths (41), possibly related to off-target adverse effects. Dalcetrapib showed less
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potent HDL cholesterol raising efficacy but was neutral with respect to CVD outcomes (42). Apparently similar neutral results were seen with evacetrapib despite an even greater mean HDL cholesterol
increase (43). While the aggregate of results raise doubts and questions about the HDL hypothesis (44),
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the lipid community awaits the report of CVD outcomes in an RCT of the last standing CETP inhibitor,
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namely anacetrapib, which also reduces LDL cholesterol; these are anticipated in the next year (45).
Familial combined hypolipidemia and evinacumab
Familial combined hypolipidemia provided another promising target for biologic therapy, leading to the development of evinacumab. Familial combined hypolipidemia is a relatively recently
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recognized autosomal recessive disorder characterized by globally reduced levels of LDL and HDL cholesterol and triglyceride, with no apparent adverse effects. The gene was found by screening families that appeared to have heterozygous FHBL, but without a mutation in the APOB gene. The hypolipidemia
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phenotype is caused by rare loss-of-function mutations affecting the ANGPTL3 gene encoding angiopoietin like protein 3 (46). ANGPTL3 is a glycoprotein that reversibly inhibits lipoprotein lipase (LPL),
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which is responsible for the hydrolysis of triglycerides within lipoproteins and clearance of chylomicrons and VLDL from blood; it also inhibits endothelial lipase, which hydrolyzes HDL (47). These mechanisms are consistent with the previous findings that gain-of-function experiments with ANGPTL3 increased total cholesterol and triglyceride levels in mice, while loss-of-function mutations were found in those in the lowest quartile of triglycerides in the Dallas Heart Study (48). Evinacumab (REGN1500, Regeneron), a human monoclonal antibody that inhibits ANGPTL3 is currently undergoing a phase 2 trial in patients with HoFH. Preliminary results reported in June 2016 showed dramatic LDL cholesterol reductions when
ACCEPTED MANUSCRIPT 11 evinacumab was added to existing therapy in three HoFH patients enrolled, with no serious adverse events identified except for injection site reactions (49). There is also some reason to believe the agent
development.
Apolipoprotein C-III deficiency and volanesorsen
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would be effective in severe hypertriglyceridemia. Anti-sense therapies against ANGPTL3 are also in
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The apparently successful application of the strategy to identify drug targets based on favorable clinical phenotypes in carriers of inactivated gene products is further exemplified by apo C-III deficiency.
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The APOC3 gene has been known to be a key determinant of plasma lipids for > 30 years (50). A study in a Pennsylvania Amish population found that carriers of a rare APOC3 gene knock-out variant had favorable lipids and decreased atherosclerosis (51). These findings were extended in two epidemiologic studies using DNA sequencing in populations under a Mendelian randomization framework, which both
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found that carriers of rare DNA variants encoding inactivated apo C-III had improved lipids and decreased lifetime CVD risk (52-53). Both monoclonal antibodies and anti-sense RNA strategies were rapidly pursued in an effort to simulate the effects of genetically knocked down apo C-III. The agent that
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is furthest along the development pathway is the anti-sense RNA drug volanesorsen (Ionis/Akcea), which in two reported early phase trials has been shown to have a potent effect on reducing plasma lipids,
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particularly severely elevated triglyceride levels (54-55). Concerns include injection site reactions and effects on hematologic variables. Whether such treatment could be applied more widely to reduce CVD risk in at-risk patients with mildly to moderately elevated lipids, already treated on a statin will certainly require definitive outcomes studies.
Human gene therapy for severe dyslipidemia
ACCEPTED MANUSCRIPT 12 Most examples above illustrate the approach of finding human deficiency states that are associated with favorable clinical phenotypes and then developing pharmacologic agents to mimic these beneficial genetic effects. The reasoning behind this approach is that typically it is easier to
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pharmacologically inhibit or disrupt a target molecule, rather than selectively boost or stimulate its activity. With targeted biologics, such as monoclonal antibodies or antisense strategies, it becomes even more feasible to precisely target the specific gene or gene product that is indicated to be clinically
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relevant by the human genetic deficiency state.
However, there are at least two examples where a human deficiency that results in a clinically
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deleterious phenotype can be corrected at least somewhat by replacing the deficient gene, although space does not permit comprehensive discussion here. First, HoFH, as mentioned above, results from virtually complete deficiency of LDL receptor activity. Promising studies of adeno-associated virus-based gene therapy in this condition in primate models (56) have resulted in accelerated development of this
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type of investigational treatment in humans (AAV8.TBG.hLDLR; RegenXbio), to be administered by intravenous infusion. Second, familial lipoprotein lipase (LPL) deficiency is an ultra-rare disorder (probably < 100 Canadian patients) due to bi-allelic mutations that abolish enzyme function and result in
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profound hypertriglyceridemia with high risk of death from pancreatitis (57). Alipogene tiparvovec (Glybera, uniQure BV) is an adeno-associated virus gene therapy that was developed to treat patients
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with LPL deficiency. It is administered intramuscularly and has recently shown some evidence of long term benefit (58). Glybera has been approved since 2012 in Europe for treatment of this condition, but has not yet been approved in North America.
Enzyme replacement in lysosomal acid lipase deficiency Lysosomal acid lipase deficiency (LALD) is an ultra-rare disorder (probably < 100 Canadian patients) due to bi-allelic mutations in the LIPA gene encoding lysosomal acid lipase (also known as
ACCEPTED MANUSCRIPT 13 cholesterol ester hydrolase). The mutations abolish function of the enzyme, resulting in combined dyslipidemia and liver injury due to cholesterol ester accumulation (59). While gene therapy is one theoretical approach to replace the missing enzyme, in this condition, the actual enzyme itself was
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developed as a drug (sebelipase alfa, Synageva) to be given by intermittent intravenous infusions. A recent phase 3 clinical trial conducted in 66 patients with LALD showed significant improvement in transaminases and reduction in hepatic lipid content (60). Kanuma (Alexion) was approved in 2015 by
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the US Food and Drug Administration and the European Medicines Agency for treatment of LALD, but has not yet been approved in Canada. Cost is an important consideration, as it is with all orphan drugs
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for ultra-rare conditions.
Angiopoietin like protein 4: a drug target candidate recently nominated by genetics Inactivating DNA variants in the ANGPTL4 gene that encodes angiopoietin like protein 4 were
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recently reported to be associated with a favorable lipid profile, including reduced triglycerides and raised HDL cholesterol together with reduced CVD risk in epidemiological samples (61-62) and also in patients ascertained through electronic health records (63). However, before jumping on the
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bandwagon to develop agents to inhibit ANGPTL4, it is worth recalling that early animal studies with a monoclonal antibody to ANGPTL4 were associated with development of intestinal lipomatosis and
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granulomatosis (64), which were also observed more recently when mice and monkeys were treated with a similar antibody (63). This example illustrates how knowledge from other non-genetic biological data needs to be integrated into the decision-making process when gauging the potential value of drug targets identified using purely genetic approaches (48).
Apolipoprotein A-V: implicated by genetics
ACCEPTED MANUSCRIPT 14 Apo A-V encoded by the APOA5 gene modulates the metabolism of triglyceride rich lipoproteins. A gargantuan DNA sequencing study found that carriers of rare inactivating APOA5 variants had ~2.2fold increased risk of early myocardial infarction, explaining ~1-2% of total cases (65). APOA5 thus
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represents another potential drug target (66), but the challenge here is that the favorable clinical
phenotype requires increased expression of the protein. A new drug would thus need to selectively boost or stimulate apo A-V production, which is more challenging to accomplish than knocking it down.
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In addition, except in ultra-rare cases of complete deficiency of apo A-V associated with severe
hypertriglyceridemia (58), most "garden variety" cases from the Mendelian randomization studies are
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heterozygous (65), with presumably only relative deficiency of apo A-V. Furthermore, plasma levels of apo A-V cover a very wide range in the "healthy" general population (66), so the therapeutic goal or target level from upregulating the gene product is uncertain at best.
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Identification of other lipid-based drug targets
We have described several examples where human genetics has pointed the way - or is pointing the way - to potentially useful new therapeutics focused on lipids that may eventually have a beneficial
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impact on hard clinical outcomes. These were largely based on rare monogenic variants (67). However, in addition to these targets, other types of genetic studies (3) have identified a plethora of additional
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variants, of which at least some might be potential targets for further consideration and development. For instance, genome-wide association studies (GWAS) of common genetic variants determining
plasma lipid levels in the general population have identified 157 highly significant gene loci out of millions that were tested (68-69). Many of these significant loci are already familiar determinants, including LDLR, HMGCR, APOB, PSCK9, ANGPTL3, LPL, APOA5, LPA and CETP, but more than half of them are within gene loci that are poorly characterized. An optimistic view is that some of the novel "hits"
ACCEPTED MANUSCRIPT 15 from GWAS will turn out to be clinically relevant. A harbinger of this relevance is that lipid-related loci represent ~ 25% of all significantly associated genes from GWAS of coronary artery disease (70).
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Conclusions
After more than a decade characterized by few new developments, and mainly by genericization of statins, the past 18 months has witnessed a re-energization of the lipid field, with approval of PCSK9
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inhibitors and some other specialty drugs for management of dyslipidemia in particular contexts.
Whether the PCSK9 inhibitors ever become more widely used will depend on results of upcoming end
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point studies. But perhaps equally striking is that there is now a rich development pipeline for even newer agents that target lipid-related genes and gene products, which originally came to attention through the study of human genetics (71). Some of these newer agents may only have niche or orphan indications. But the same could have been said of statins 30 years ago, when their initial use was
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restricted to lipid and metabolic clinics, but through medical progress and mounting clinical trial evidence, spread to the general cardiology population. At least four lipid-lowering agents mentioned in the 2016 Canadian Lipid Guidelines - statins, ezetimibe, PSCK9 inhibitors, and lomitapide (72) - have
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development stories that are traceable to human genetics, with perhaps more examples pending.
ACCEPTED MANUSCRIPT 16 Acknowledgements/disclosures RAH is supported by the Jacob J. Wolfe Distinguished Medical Research Chair, the Edith Schulich Vinet
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Research Chair in Human Genetics, and the Martha G. Blackburn Chair in Cardiovascular Research.
RAH has received operating grants from the Canadian Institutes of Health Research (Foundation Grant), the Heart and Stroke Foundation of Ontario (T-000353), and Genome Canada through Genome Quebec
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(award 4530).
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RAH has received honoraria for membership on advisory boards and speakers' bureaus for Aegerion, Amgen, Gemphire, Lilly, Merck, Pfizer, Regeneron, Sanofi and Valeant.
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LRW has no disclosures to report.
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ACCEPTED MANUSCRIPT 19 27. Sacks FM, Stanesa M, Hegele RA. Severe hypertriglyceridemia with pancreatitis: thirteen years' treatment with lomitapide. JAMA Intern Med 2014; 174:443-7. 28. Burnett JR, Hegele RA. Finding the Therapeutic Sweet Spot: Using Naturally Occurring Human Variants to Inform Drug Design. Circ Cardiovasc Genet 2015; 8:637-9.
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39. Voight BF, Peloso GM, Orho-Melander M, et al. Plasma HDL cholesterol and risk of myocardial infarction: a Mendelian randomisation study. Lancet 2012; 380:572-80. 40. Wu Z, Lou Y, Qiu X, et al. Association of cholesteryl ester transfer protein (CETP) gene polymorphism, high density lipoprotein cholesterol and risk of coronary artery disease: a meta-analysis using a Mendelian randomization approach. BMC Med Genet 2014; 15:118.
ACCEPTED MANUSCRIPT 20 41. Barter PJ, Caulfield M, Eriksson M, et al. Effects of torcetrapib in patients at high risk for coronary events. N Engl J Med 2007; 357:2109-22. 42. Schwartz GG, Olsson AG, Abt M, et al. Effects of dalcetrapib in patients with a recent acute coronary syndrome. N Engl J Med 2012; 367:2089-99.
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ACCEPTED MANUSCRIPT 22 71. Kathiresan S. Developing medicines that mimic the natural successes of the human genome: lessons from NPC1L1, HMGCR, PCSK9, APOC3, and CETP. J Am Coll Cardiol 2015; 65:1562-6. 72. Anderson TJ, Grégoire J, Pearson GJ, et al. 2016 Canadian Cardiovascular Society Guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult. Can J Cardiol
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23
MTTP
1990
Hypobetalipoproteinemia
APOB
1983
Proprotein convertase subtilisin kexin 9 (PSCK9) deficiency
PCSK9
2003
Cholesteryl ester transfer protein (CETP) deficiency
CETP
1989
Familial combined hypolipidemia
ANGPTL3
2001
Apolipoprotein C-III deficiency
APOC3
Lipoprotein lipase deficiency
LPL
Elevated LDL cholesterol Depressed apo Bcontaining lipoproteins Depressed apo Bcontaining lipoproteins Low LDL cholesterol
Elevated HDL cholesterol; low LDL cholesterol Low LDL cholesterol and triglycerides Low triglycerides and elevated HDL cholesterol Elevated triglycerides
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1985
Stage of development Approved worldwide between 1987 and 2001 (pitavastatin not approved in Canada). Now all are genericized in Canada. They are the standard of care in CVD prevention.
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1981
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LDLR
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Familial hypercholesterolemia Abetalipoproteinemia
Resulting therapeutic(s) statins (lovastatin, pravastatin, simvastatin, fluvastatin, atorvastatin, rosuvastatin, pitavastatin) LDL receptor gene therapy lomitapide (Juxtapid)
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Table 1. Lipid therapies that can be traced to study of familial disorders Rare familial disorder Implicated Approximate year Biochemical gene name of gene cloning* phenotype Familial LDLR 1981 Elevated LDL hypercholesterolemia cholesterol
mipomersen (Kynamro)
Phase 2 clinical trials underway in 2016. Approved in the US and Europe in 2013, and in Canada in 2014 for homozygous familial hypercholesterolemia (Juxtapid). Approved in US only in 2013 for homozygous familial hypercholesterolemia (Kynamro).
evolocumab (Repatha), alirocumab (Praluent), bococizumab anacetrapib
Approval worldwide as of 2015. In Canada evolocumab (Repatha) approved in 2015 and alirocumab (Praluent) approved in 2016. Development of bococizumab terminated in November 2016 due to safety concerns. Not yet approved. Phase 3 clinical trials since 2003.
evinacumab
Not yet approved. Phase 2/3 clinical trials since 2015.
volanesorsen
Not yet approved. Phase 2/3 clinical trials since 2013.
alipogene Approved in Europe in 2012 (Glybera). tiparvovec (Glybera) * or linkage of the gene to the human disease phenotype; abbreviations: LDL, low density lipoprotein; apo, apolipoprotein; CVD, cardiovascular disease
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24 Table 2. Examples of lipid drug targets that have been validated in Mendelian randomization studies Lipid effect
HMGCR
Loss-of-function: lowers LDL cholesterol (also increases weight and diabetes risk)
PSCK9
Loss-of-function: lowers LDL cholesterol
Loss-of-function: lowers CHD risk
NPC1L1
Loss-of-function: lowers LDL cholesterol
Loss-of-function: lowers CHD risk
APOC3
Loss-of-function: lowers TG, raises HDL cholesterol Loss-of-function: lowers TG, raises HDL cholesterol
Loss-of-function: lowers CHD risk Loss-of-function: lowers CHD risk
Loss-of-function: raises TG, lowers HDL cholesterol Gain-of-function: raises Lp(a) levels
Loss-of-function: increases CHD risk Gain-of-function: increases CHD risk
LPA
Inhibitors lower both LDL cholesterol and CHD risk in virtually all statin trials in both primary and secondary prevention. Inhibitors lowers both LDL cholesterol and CHD risk in metaanalysis of phase 3 studies. Inhibitor lowers both LDL cholesterol and CHD risk on top of statin (e.g. ezetimibe in the IMPROVE-IT study). Inhibitor lowers TG levels; CHD study not done yet. Inhibitor improves lipid profile, but may have off-target effects; CHD study not done yet. Not studied yet.
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APOA5
CHD effect in clinical trials
Inhibitor reduces Lp(a) levels selectively, CHD study not done yet.
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ANGPTL4
CHD effect in genetic studies Loss-of-function: lowers CHD risk
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Target
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abbreviations: CHD, coronary heart disease; LDL, lower density lipoprotein; TG, triglyceride