Disclosures

Scientific Advisory Board Research Grants

Regeneron Genetics Center Aegerion

Eli Lilly

Merck

Merck Bayer Healthcare

Novartis Regeneron

Amarin

Celera

Developing medicines that mimic the natural successes of the genome

Sekar Kathiresan, MD Director, Preventive Cardiology, MGH

Associate Professor of Medicine, Harvard Medical School Institute Member, Broad Institute

ISA 2015

May 24, 2015

http://www.cdc.gov/nchs/data/dvs/deaths_2010_release.pdf

Myocardial infarction: ���leading cause of death in US and worldwide

Major unmet need: ���only 2 therapies to prevent FIRST MI & ���

both with modest efficacy ���

•  Aspirin •  Statins

Prevents clotting Reduces relative risk by ~15%

Lowers LDL Reduces relative risk by ~25%

Problem:

Only about 5% of medicines in development succeed into clinic

Two reasons stand out:

Poorly predictive models

Two reasons stand out:

Poorly predictive models Don’t know impact of blocking a gene over

many years

Idea:

Identify naturally-occurring mutations that protect against disease &

develop medicines that mimic them

MI

Medicines that mimic natural successes of the genome

MI

Initial example for MI

PCSK9

1 in 50 blacks

Lower LDL

80% lower risk

alirocumab, evolocumab, bococizumab

Abifadel, Nat Genet (2003)

Cohen, N Engl J Med (2006)

Stein, N Engl J Med (2012)

MI

Are there more examples for MI?

Outline

Results: Risk Mutations

LDLR, APOA5 Protective Mutations

NPC1L1, LPA LPL, APOC3

Two Human Genetics Experiments

Experimental Approach

Outline

Results: Risk Mutations

LDLR, APOA5 Protective Mutations

NPC1L1, LPA LPL, APOC3

Two Human Genetics Experiments

Experimental Approach

Ascertain MI cases & controls

Cases with MI Controls free of MI

Characterize genome in each person for NULL mutations

Variant in Exon

AATCATCGATGT

AATCATTGATGT

Synonymous

Missense

Nonsense

Splice site Null mutations

Indel frameshift

Normal: 2 working copies of a gene

With null mutations: ���one or two copies of a gene broken

Heterozygous null Homozygous null

Why focus on null mutations?���

Heterozygous null

Most therapies inhibit target gene

(rather than activate target)

Test if carriers of null mutations in a given gene are protected against MI

Cases with MI Controls

Cases Controls

Cumulative frequency of

null mutations in gene

X %

Y %

Protective signal: frequency of null mutations greater in disease-free controls than cases

Cases with MI Controls

Cases Controls

Cumulative frequency of

null mutations in gene

X %

Y %

Risk gene

X > Y

Protective gene

Y > X

Outline

Results: Risk Mutations

LDLR, APOA5 Protective Mutations

NPC1L1, LPA LPL, APOC3

Two Human Genetics Experiments

Experimental Approach

Study design: MI/CHD cases & controls

Exome Sequencing

Cases N=5K

Controls N=5K

Cases N=60K

Controls N=120K

Exome Chip Genotyping

Do et al., Nature 2015

Stitziel et al., Under Review

Systematically search for Protective Mutation signal across each of ~18,000 genes

Outline

Results: Risk Mutations

LDLR, APOA5

Two Human Genetics Experiments

Experimental Approach

Protective Mutations NPC1L1, LPA LPL, APOC3

 

Early MI risk mutation signal for ���each of 18,000 genes

One gene stands out

Low-density lipoprotein receptor (LDLR)

Do et al., Nature (2015)

Clinical Effect

Low-density lipoprotein receptor (LDLR)

1 in 217 carrier  

 

Loss of function mutations at LDLR lead to familial hypercholesterolemia (FH) & increase MI risk

LDL +75%

MI

4-fold

Gene

Where does this oft-quoted come from?

Textbooks: FH carrier frequency is 1 in 500

Nordestgaard, Eur Heart J (2013)  

Goldstein, J Clin Invest 52:1544, 1973

Patients with early-onset MI Men or women <60y

Controls without MI

1 in 500 with total cholesterol > 285 mg/dl

In 1973, Goldstein defined FH as TC > 285 mg/dl

0.2% = 1 in 500

Goldstein, J Clin Invest 52:1544, 1973

Patients with early-onset MI Controls without MI

LDLR mutations in cases: 1 in 51

LDLR mutations in controls: 1 in 217

In sequencing era, we (and others) have refined this estimate to

~ 1 in 200

Do et al., Nature (2015)

Clinical Effect

Low-density lipoprotein receptor (LDLR)

1 in 217 carrier  

 

Loss of function mutations in APOA5 also increase MI risk [as well as plasma triglyceride-rich lipoproteins (TRL)]

Apolipoprotein A5 (APOA5)

LDL +75%

MI

4-fold

1 in 158 carrier  

 

Gene

TRL

+60% MI

2-fold

Outline

Results: Risk Mutations

LDLR, APOA5

Two Human Genetics Experiments

Experimental Approach

Protective Mutations NPC1L1, LPA LPL, APOC3

 

NPC1L1���Niemann-Pick C1-Like 1

Clinical question

Will addition of an LDL-lowering medicine on top of statin

improve clinical outcomes?

Ezetimibe (Zetia) lowers LDL by blocking cholesterol absorption by 50%

•  Ezetimibe targets NPC1L1 gene in the intestine

Francis, CMAJ (2005)

Acetyl CoAyy

Cholesterol biosynthesisol bol b

Bile acid biosynthesis

Bile acids

Bile

Bile acids

Enterohepaticcirculation

Terminal ileum

Sterols

Bile acids

Diet

Upper small intestine

Lipoprotein synthesis

LDL receptorLLDL

Plasmacompartment

Liver

Lymphatics

Chylomicrons

Cholesterol

s

Ezetimibe

Statins

It was uncertain if lowering LDL through ezetimibe would

reduce risk for MI

Human genetics experiment

to address this issue

NPC1L1 null mutations and ezetimibe both reduce protein function by ~50%

Genetics

Pharmacology

Heterozygous null – protein function reduced

lifelong by 50%

Ezetimbe treatment reduces cholesterol absorption by 54%

Sudhop, Circulation (2002)

Genetics mimics pharmacology

People who carry NPC1L1 null mutations:

it’s as if they had been given ezetimibe lifelong

Our two hypotheses:

When compared with non-carriers, carriers of NPC1L1 null mutations will have:

1.  lower LDL cholesterol

2.  lower risk for MI

NPC1L1 null mutations: nonsense, splice, frameshift���seen in ~ 1 in 650 in the population

NPC1L1 null mutation carriers ���have lower LDL

- 12 mg/dl

LDL

NPC1L1 null mutation carriers have lower LDL and protected against MI

- 12 mg/dl

- 53%

LDL MI

Nate Stitziel, MD, PhD Hong-Hee Won, PhD

N Engl J Med Epub 2014 Nov 12  

These genetic results predict that ezetimibe should reduce MI risk

…a week later,

at American Heart Association 2014

IMProved Reduction of Outcomes: Vytorin Efficacy International Trial A Multicenter, Double-Blind, Randomized Study to Establish the Clinical Benefit and Safety of Vytorin (Ezetimibe/Simvastatin Tablet) vs Simvastatin Monotherapy in High-Risk Subjects Presenting With Acute Coronary Syndrome

Patients stabilized post ACS ≤ 10 days: LDL-C 50–125*mg/dL (or 50–100**mg/dL if prior lipid-lowering Rx)

Standard Medical & Interventional Therapy

Ezetimibe / Simvastatin 10 / 40 mg

Simvastatin 40 mg

Follow-up Visit Day 30, every 4 months

Duration: Minimum 2 ½-year follow-up (at least 5250 events)

Primary Endpoint: CV death, MI, hospital admission for UA, coronary revascularization (≥ 30 days after randomization), or stroke

N=18,144

Uptitrated to Simva 80 mg if LDL-C > 79 (adapted per

FDA label 2011)

Study Design *3.2mM

**2.6mM

Cannon CP AHJ 2008;156:826-32; Califf RM NEJM 2009;361:712-7; Blazing MA AHJ 2014;168:205-12

90% power to detect ~9% difference

LDL-C and Lipid Changes 1 Yr Mean LDL-C TC TG HDL hsCRP Simva 69.9 145.1 137.1 48.1 3.8

EZ/Simva 53.2 125.8 120.4 48.7 3.3

Δ in mg/dL -16.7 -19.3 -16.7 +0.6 -0.5

Median Time avg 69.5 vs. 53.7 mg/dL

Primary Endpoint — ITT

Simva — 34.7% 2742 events

EZ/Simva — 32.7% 2572 events

HR 0.936 CI (0.887, 0.988) p=0.016

Cardiovascular death, MI, documented unstable angina requiring rehospitalization, coronary revascularization (≥30 days), or stroke

7-year event rates

NNT= 50

Compare genetics vs pharmacology

- 12 mg/dl

- 53%

LDL MI

- 15 mg/dl - 6.4%

LDL MI

NPC1L1 null mutations

Ezetimibe

Hypothesis: lifelong lower LDL ���amplifies effect on MI

- 12 mg/dl

- 53%

LDL MI

- 15 mg/dl - 6.4%

LDL MI

Ezetimibe NPC1L1 null mutations

“You can never be too rich or have too low an LDL.”

…Eugene Braunwald, MD

LPA���Lipopoproetin (a)

Null mutations at LPA lower Lp(a) and reduce MI risk

Ogorelkova, Gruber, Uttermann, Hum Mol Genet (1999) Lim et al., PLoS Genetics (2014)

Lipoprotein (a) (LPA)

1 in 13 carrier  

 

Gene Null mutations

c.4974-2A>G & c.4289+1G>A

Clinical Effect

Lp(a) MI

-60% -14%

Outline

Results: Risk Mutations

LDLR, APOA5

Two Human Genetics Experiments

Experimental Approach

Protective Mutations NPC1L1, LPA LPL, APOC3

 

LPL ���Lipoprotein lipase

Wittrup et al., Circulation (1999) Varbo et al., J Am Coll Cardiol (2013) Stitziel et al., under review

Lipoprotein lipase (LPL)

1 in 10 carrier  

 

Null mutation at LPL lowers TRL, increases HDL, and reduces MI risk

Gene Null mutations

S447X

Clinical Effect

TRL MI

-10% -17%

 APOC3���

Apolipoprotein C-III    

Crosby et al. N Engl J Med (2014)

Clinical Effect Apolipoprotein C-III (APOC3)

TRL MI

-39%

IVS1-2GàA IVS2+1GàA

R19X

1 in 150 individuals are heterozygous for

null mutations

     

Apolipoprotein C-III gene: ���Heterozygous null mutations lower triglycerides

Crosby et al. N Engl J Med (2014)

TRL MI

-39% -40%

IVS1-2GàA IVS2+1GàA

R19X

1 in 150 individuals are heterozygous for

null mutations

     

Apolipoprotein C-III gene: ���Heterozygous null mutations protect against MI

Clinical Effect Apolipoprotein C-III (APOC3)

These genetic results predict that inhibition of APOC3 should reduce MI risk

TG

- 43%

•  Administration of therapeutic to healthy human volunteers

•  APOC3 level by 70%

Graham, Circ Res (2013)  

TG

- 43%

MI ? •  Administration of therapeutic to healthy human volunteers

•  APOC3 level by 70%

Graham, Circ Res (2013)  

Why are APOC3 mutation ���carriers protected?

People WITHOUT mutation (RR)

People WITH mutation (RX)

Oral fat challenge test��� in mutation non-carriers and carriers

People WITHOUT mutation (RR)

People WITH mutation (RX)

Dietary fat challenge

After a high-fat meal, ���plasma triglycerides normally increase

Pollin, Science 2008  

In APOC3 null mutation carriers, ���triglycerides increase is markedly blunted

Pollin, Science 2008  

Now, six protective mutation examples for MI

PCSK9

NPC1L1

LPA

LPL

APOC3

ANGPTL4

1 in 50 blacks 1 in 150 1 in 13 1 in 10 1 in 150 1 in 500

LDL LDL Lp(a) TRL TRL TRL

80% lower risk

53% lower risk

14% lower risk

17% lower risk

40% lower risk

57% lower risk

alirocumab, evolocumab, bococizumab

ezetimibe Antisense in development ?

Antisense in development

Monoclonal antibodies in development

MI

Take Home #1: Multiple genes suggest ���post-prandial lipoprotein metabolism ���

as key mechanism for atherosclerosis (beyond LDL)

Zilversmit, Circulation (1979)

Post-prandial TRL metabolism genes ���with compelling human genetic evidence

Lipoprotein Lipase

APOA5

APOC3 ANGPTL4 ANGPTL3

Mutations in all five genes affect plasma TRL,���in 4 affect MI risk,���

3 with protective null alleles

Lipoprotein Lipase

APOA5

APOC3 ANGPTL4 ANGPTL3

HDL

LDL

IDL VLDL Chylomicron Remnant

apo A-I apo B apo B apo B apo B48

Cholesterol Triglyceride

Take Home #2: human genetic studies uniformly point to apoB-containing lipoproteins as

key drivers of atherosclerosis

Lp(a)

apo B

apo(a)

Triglyceride-rich lipoproteins (TRLs)

Leverage naturally-occurring variation in target genes to anticipate

efficacy (and side effects) of medicines

Acknowledgments Kathiresan laboratory Broad Genomics Platform

Stacey Gabriel, PhD

Collaborators

Funding

MGH: Research Scholar Award