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JLRInvitedLp(a)ThematicReviewSeriesContribution

AntisenseInhibitionofApolipoprotein(a)toLowerPlasma

Lipoprotein(a)LevelsinHumans

MarkJ.Graham1,NickViney1,RosanneM.Crooke1,SotiriosTsimikas1,2

1IsisPharmaceuticalsand2DivisionofCardiovascularMedicine,UniversityofCaliforniaSan

Diego,LaJolla,CA.

Wordcount:7502

Correspondenceto:SotiriosTsimikas,UniversityofCaliforniaSanDiego,BSB-1080,

LaJolla,CA,92093-0682,stsimikas@ucsd.edu

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Abstract

Epidemiologic,geneticassociationandmendelianrandomizationstudieshaveprovided

strongevidencethatlipoprotein(a)[Lp(a)]isanindependent,causalriskfactorfor

cardiovasculardisease(CVD),includingmyocardialinfarction,stroke,peripheralarterial

disease,andcalcificaorticvalvestenosis.Lp(a)levels>50mg/dLarehighlyprevalent(20%of

thegeneralpopulation)andareoverrepresentedinpatientswithCVDandaorticstenosis.

ThesedatasupportthenotionthatLp(a)shouldbeatargetoftherapyforCVDeventreduction

andtoreduceprogressionofaorticstenosis.However,effectivetherapiestospecificallyreduce

plasmaLp(a)levelsarelacking.RecentanimalandhumanstudieshaveshownthatLp(a)canbe

specificallytargetedwithsecondgenerationantisenseoligonucleotides(ASOs)thatinhibit

apo(a)mRNAtranslation.Inapo(a)transgenicmice,anapo(a)ASOreducedplasmaapo(a)/Lp(a)

levelsandtheirassociatedoxidizedphospholipidlevelsby86%and93%,respectively.In

cynomolgousmonkeys,asecondgenerationapo(a)ASO,ISIS-APO(a)Rx,significantlyreduced

hepaticapo(a)mRNAexpressionandplasmaLp(a)levelsby>80%.Finally,inaPhaseIstudyin

normalvolunteersISIS-APO(a)RxASOreducedLp(a)levelsandtheirassociatedoxidized

phospholipidlevelsupto89%and93%,respectively,withminimaleffectsonother

lipoproteins.ISIS-APO(a)Rxrepresentsthefirstspecificandpotentdruginclinicaldevelopment

tolowerLp(a)levelsandmaybebeneficialinreducingCVDeventsandprogressionofcalcific

aorticvalvestenosis.

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Introduction

Lipoprotein(a)[Lp(a)]isahighlypolymorphiclipoproteinfoundinhumanplasmain

levelsrangingfrom<1mg/dLto>250mg/dL.Lp(a)consistsofanLDL-likeparticleand

apolipoprotein(a)[apo(a)],whicharecovalentlyboundviaadisulfidebondbetweenCys4326of

apoB-100andCys4057ofapo(a)locatedinkringlefourtype9(KIV9).Apo(a)comprisestenKIV

subunits,ofwhichKIV2ispresentinvariable,identically-sizedrepeats,KVandaninactive

proteasedomain.Theapo(a)proteinshowsahighdegreeofhomology(75-100%)to

plasminogenatboththenucleotideandtheaminoacidlevel(1).However,theapo(a)gene

transcriptismuchlargerduetotherepetitiveKIV2domain(3to>40repeats)intheLPAgene.

Themajorityofapo(a)mRNAisexpressedintheliver,withminoramountspresentinthetestis,

brain,adrenals,lung,andpituitary.Lp(a)levelsareprimarilygeneticallydeterminedbytheLPA

allelespresentwithinanindividual(2,3).

Epidemiologic,geneticassociationandmendelianrandomizationstudieshaveprovided

strongevidencethat[Lp(a)]isanindependent,causalriskfactorforcardiovasculardisease

(CVD),includingmyocardialinfarction,stroke,andperipheralarterialdisease,aswellascalcific

aorticvalvestenosis(4-9).Whileextensiveepidemiologicaldatasuggeststhatelevatedplasma

Lp(a)levelsarepro-atherogenic(10),themolecularmechanismsbywhichitcontributestothe

atheroscleroticprocessremainenigmatic.Thereisevidencethatapo(a)potentiates

atherothrombosisthroughitsLDLmoietyandbyadditionalapo(a)-drivenmechanisms,

includingimpairingfibrinolysis,mediatingpro-inflammatoryeffects,activatingendothelialcells,

recruitingmonocytes,acceleratingmacrophagefoamcellformationandtransportingpro-

inflammatoryoxidizedphospholipids(1,11-16).

DuetotheexpandingevidenceindicatingthatLp(a)contributestoCVD,asearchfor

potentandspecificinhibitorsofapo(a)wasinitiated.Amongthevarioustherapeuticplatforms

amenabletoselectiveinhibitionofLp(a),antisenseoligonucleotide(ASO)drugshaveemerged

asapromisingapproachinloweringLp(a)levelsintheclinicalsetting.Antisense

oligonucleotidesrepresentthethirdmajortherapeuticdiscoveryplatform,distinctfromsmall

moleculeandmonoclonalantibodyapproaches,andhaveshowngreatpromiseinspecific

targetingofdisease-associatedgenesindyslipidemia,oncology,neurologicaldysfunctionand

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metabolicdisorders.Duetotheirmodeofaction,bybindingcomplementarymRNAtargetsvia

Watson-Crickbasepairing,isoform-specifictargetingispossible.InthecaseofLp(a),thisvery

importantfeatureallowsdirecttargetingoftheapo(a)transcriptwithoutalteringplasminogen

transcriptlevels

Antisenseoligonucleotidestargetingapo(a)

SecondgenerationASOsaresingle-stranded,chimericmoleculesgenerally20

nucleotidesinlength,containing2ʹ-O-(2-methoxyethyl)[MOE]modificationsat5ʹand3ʹtermini

(positions1–5and16–20)andDNA-likenucleotidesinthecentralregion(position6-15)witha

phosphorothioate(P=S)backbonethroughouttoenhancenucleaseresistance(17-19).These

moleculesareupto15-foldmorepotentthanfirst-generationP=SonlymodifiedASOdrugs,

duetotheirenhancedmRNAaffinityviatheMOEmoiety(19-21)theirgapmerdesign

supportinganRNaseH1enzymaticterminationmechanismandtheirimproved

pharmacokineticpropertiesthatpermitweekly,monthlyorpotentiallyquarterlydosing(Figure

1).Thesedrugsalsohaveanimprovedtherapeuticindexduetoreducedpro-inflammatory

properties(22-26).

SinceASOdrugsaremetabolizedbycellularnucleasesandnotthecytochromeP450

system,theycanbesafelyco-administeredwithtraditionaltherapeuticagentswithdiffering

modesofaction(20).Additionally,astheyarehydrophilic,theymaybeadministeredinsaline

withoutspecialformulationviasubcutaneous,intravenous,topical,aerosol,enema,

intravitreal,intraventricular,intrathecalandoralroutes(27).Thepharmacokineticproperties

ofASOshavebeenextensivelyquantifiedinmultiplespeciesandinman(22).Following

systemicadministration,theliver,kidney,bonemarrow,adiposetissue,spleen,andlymph

nodesaccumulatethehighestdrugconcentrations,whiledistributingpoorlytointestine,

skeletalmuscle,heart,lung,reproductiveorgans,pancreas,andbrain.MOEmodifiedASOsare

resistanttoexonucleasedegradation,resultinginprolongedtissuehalf-lives,rangingfrom10to

30days.Ingeneral,ASOareclearedfromtissuebyendonucleolyticdegradation,producing

lowermolecularweightmetabolites(8–12nucleotides)thatareeliminatedbyurinary

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excretion.

ReviewofAntisenseStudiesReportingLp(a)Levels

Inthefirstdescribedantisensestudyperformedinvitro,ahumanapo(a)ribozyme

oligonucleotidecontainingphosphorothioateDNAandRNAwasdesignedtospecificallytarget

withkringle4oftheapo(a)mRNAwithoutalteringplasminogentranscriptlevels(28).Because

humancelllinesdonotadequatelyexpresstheapo(a)gene,anapo(a)expressionvector

containingthe5’-untranslatedregion,thesignalsequence,thefirst5kringle4–likerepeats,and

291bpofthesizekringlerepeatofapo(a)drivenbythecytomegaloviruspromoterwasco-

transfectedintoHepG2cellswithaHVJ-liposome–apo(a)DNAcomplexandeithertheapo(a)

ribozymeorcontrololigonucleotide.After72hr,theapo(a)ribozymewasshowntoreduce

HepG2proteinsecretionbyapproximately60%whilenosignificantchangeinplasminogen

proteinwasobserved.Whiletheseinitialresultswereencouraging,therequirementof

liposomalformulationoftheapo(a)targetingribozymepriortodeliverywouldhavemadein

vivopharmacologystudiesmorechallenging.

Mipomersen,asecondgenerationASOdirectedtoapoB-100andapprovedforclinical

useintheUnitedStatesforloweringLDL-CinpatientswithhomozygousFamilial

Hypercholesterolemia(29),hasbeenshowntolowerLp(a)levelsinLp(a)-transgenicmice(30)

andinhumans(31-34).Inhumans,fourPhase3trialswereperformedin382patientson

maximallytoleratedlipid-loweringtherapyandrandomized2:1toweeklysubcutaneous

mipomersen200mg(n=256)orplacebo(n=126)for26weeks.Mipomersenreducedplasma

Lp(a)levelsby21-39%,whereasnosignificantchangewasnotedintheplacebogroups(35).

Interestingly,inthemipomersengroup,onlymodestcorrelationswerepresentbetween

percentchangesinLp(a)andapoB(r=0.43,p<0.001)andLp(a)andLDL-C(r=0.36,p<0.001)

suggestingmechanismsofLp(a)loweringrelatedtoliversynthesisofapoBthatarenot

apparentbyevaluatingplasmaapoBlevels.

AstudyinLp(a)-transgenicmicebyMerkietal(30)suggestedthatonepotential

mechanismofLp(a)reductionbymipomersenmaybethroughlimitinghepaticproductionof

newlyformedapoBconcomitantlywhenapo(a)isavailabletocreateanLp(a)particle.

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TransgenicmiceoverexpressingbothhumanAPOB(h-apoB-100)plushumanLPAtogenerate

genuineLp(a)particles(humanapo(a)doesnotformacovalentbondwithmouseapoB-100)

weretreatedwithmipomersen.MipomersenreducedhepaticapoBproductionandplasma

levelsofh-apoB-100toverylowlevels(<20mg/dl)andreducedLDL-CandLp(a)levelsby~75%.

However,themicecontinuedtoproducesimilaramountsofapo(a)unboundtoh-apoBas

beforetreatmentwithmipomersen,suggestingthatapoB-100synthesisisalimitingfactorin

Lp(a)particlegenerationinthisLPAtransgenicmodel(Figure2A).

Inafollow-upinvestigation,Merkietal(12)evaluatedISIS-144367,asecondgeneration

apo(a)-specificASOinthefollowingmousemodels:1-LPAtransgenicmiceexpressinga

truncatedhumanLPAcDNAwith8KIVrepeats(8K-L(a)mice)thathasveryhighLp(a)levels(11,

15,36).Thisconstructcontainsawild-typehumanapo[a]cDNAencodedKringlesIV1,IV2,a

fusionofIV3andIV5,IV6toIV10,KV,andtheprotease-likedomain.Thepromoterforthis

constructconsistsoftheapoEhepaticcontrolregionLE6andapoEintron1;2-Lp(a)-transgenic

miceexpressingbothhumangenomicAPOBandthehumanLPA(8K-Lp(a)mice);and3-LPA

transgenicmiceexpressingthenaturalhumanapo(a)genecontaining12KringleIVrepeatsina

completegenomicconstruct(12K-apo(a)mice)thatcontainsthenaturalapo(a)promoterand

regulatorysequences.AlltransgenicmiceadministeredISIS144367for4weeksshowed

significantreductionsinhepaticapo(a)mRNAexpression.ISIS144367significantlyreduced

Lp(a)by24.8%in8K-Lp(a)miceandreducedapo(a)levelsby19.2%in8K-Lp(a)mice,30.0%in

8K-apo(a)mice,and86%in12K-apo(a)mice(Figure2B).Themostpotenteffectwas

documentedin12K-apo(a)miceexpressingapo(a)withmultipleKIV2repeatscontainingthe

naturalLPApromoterandregulatorysequences.Importantly,inthe12K-apo(a)mice,ISIS

144367alsosignificantlyreducedplasmaoxidizedphospholipidsonapoB-containing

lipoproteins(OxPL-apoB)by86%.Alargenumberofclinicalstudieshavedemonstratedthat

OxPL-apoB,mainlyreflectingthebiologicalactivityofsmallapo(a)isoformswithhighLp(a)

levels,arestrongpredictorsofCVDevents,lendingevidencethatthecontentofpro-

inflammatoryOxPLarekeydeterminantsofatherogenicriskmediatedbyLp(a)(37-42).

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IdentificationofaSecondGenerationAntisenseDrugntoHumanApo(a)

Inlightofthepromisingactivityobservedinthetransgenicmousestudiesdescribed

aboveandthedesiretoidentifyanoptimizedhumanclinicalcandidate,ahighdensityscreenof

over2200secondgenerationASOsdesignedtocomplementarysitesspanningtheentire

humanapo(a)transcriptweretestedfortheirabilitytodose-dependentlyreduceapo(a)mRNA

expressionintransgenicLPAmouseprimaryhepatocytes.AnoptimalASO,nowdesignatedas

ISIS-APO(a)RxtodifferentiateitfromthepriorversionISIS144367,wasidentifiedthatbindsto

theexon24-25splicesiteofthematurehumanapo(a)transcript(GenbankAccession

NM_005577.2)atposition3901-3920bp(Table1).KIV2repeatsareidenticalattheprotein

level,butarenotconserveduniformlyatthenucleotidelevel,whichiswhythedrugonlybinds

toasinglesplicejunctionwithperfectcomplementarity.ISIS-APO(a)Rxalsohasthepotentialto

bindto11alternativesiteswithinthetranscriptcontaining1-4mismatchednucleotides,

relativetotheactivesite.TheconcentrationofISIS-APO(a)Rxthatproduceda50%reduction

(IC50)oftheapo(a)mRNAinLPAtransgenicmouseprimaryhepatocyteswas0.12μM.In

cynomolgousmonkeyprimaryhepatocytes,theobservedIC50was0.49μM.Incontrast,theIC50

valuesforcontrolASOsnottargetedtotheapo(a)mRNAwere>10μMinbothprimarycell

isolates(datanotshown).

PharmacologyofISIS-APO(a)RxinHumanLPATransgenicMice

Astheapo(a)transcriptisnotexpressedinrodents,invivopreclinicalefficacy

assessmentswerelimitedtostudiesintransgenicmiceexpressinga12kringleKIVapo(a)

isoformwhichexpressedtheentirehumanLPAgenomicsequence,withouthumanapoB(43).

AdministrationofISIS-APO(a)Rxto12K-apo(a)miceproduceddose-dependentreductionsin

apo(a)livermRNAandapo(a)inplasmaafter2weeksofASOadministrationat1.5,5,15and50

mg/kg/week(Figure3).The50%effectivedose(ED50)valuesforISIS-APO(a)Rxapo(a)mRNAand

plasmaapo(a)reductionswere9.7and12.4mg/kg/week,respectively,inthistransgenicmodel.

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PharmacologyofISIS-APO(a)RxinLeanCynomolgusMonkeys

WhiletheISIS-APO(a)Rxbindingsiteintherhesusmonkeycontainsasinglemismatch

relativetothenonhumanprimatesequence,thepotentialpharmacodynamiceffectsofthis

compoundwereevaluatedinchowfedcynomolgusmonkeyswhenadministeredupto40

mg/kg/weekfor12weeks.Cynomolgusmonkeys,inasimilarfashiontohumans,haveawide

rangeofplasmaLp(a)levelsduetovariabilityintheKIV-2repeatssimilartohumans,butlack

KVofhumanapo(a)(44).Asdescribedpreviouslyinbothhumansandnonhumanprimates,our

datashowthatcynomolgusmonkeyshadhighlyvariablehepaticmRNAexpressionlevels(44,

45).ResultsfromthisstudydemonstratedthatISIS-APO(a)Rxsignificantlyreducedhepatic

apo(a)mRNAby90%,relativetothesalineadministeredcohort(Table2).Furthermore,dueto

someconservationofapo(a)andplasminogennucleotidesequences(thereare3-base

mismatcheswithinthenearhomologousbindingsite),plasminogenmRNAlevelswerealso

measured.TherewasnosignificantchangeinhepaticplasminogenmRNAdetected,relativeto

thePBScohort,after12weekISIS-APO(a)Rxadministration(Table2).

Inanother13weekstudy,theeffectsofISIS-APO(a)Rxinhibitionasafunctionofdose

wereevaluatedinchow-fedcynomolgusmonkeys.Atthe4,8,12and40mg/kg/weekdoses,

hepaticapo(a)mRNAwasreducedto84%±12,70%±18,79%±17,and97%±3ofmeansaline

controlexpressionlevelsbyDay93ofISIS-APO(a)Rxadministration(Figure4A).Asanticipated,

plasmaLp(a)levelswereconcomitantlyreducedby23%±13,40%±22,70%±16,and90%±5

atthe4,8,12and40mg/kg/weekdoses,respectively,relativetoDay1baselinelevels(Figure

4B).Interestingly,whenplasmalipidsweremeasuredafter13weeks,therewerenosignificant

changesobservedintotalcholesterol,HDL-C,LDL-CorapoBlevelsevenatthehighest

administereddose(Table2).

Inordertoevaluatetheheterogeneityofapo(a)allelicexpressionpatternswithinthe

PBSandISIS-APO(a)Rxcynomolgusmonkeytreatmentgroups,Westernblottingwasperformed

todirectlycompareDay1(predose)andDay93samplesinbothsalineand12mg/kg/wkISIS-

APO(a)Rxcohorts(Figure5A).Theapo(a)bandintensitiesobservedwereconsistentwith

derivedplasmaapo(a)levels(Figure5B).Furthermore,asdescribedpreviously,monkeysinthis

studywerebothheterozygousandhomozygousfordifferentapo(a)isoformsizes.Importantly,

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inallfourmonkeystreatedwithISIS-APO(a)Rx,plasmaapo(a)levelswerereducedtonearly

undetectablelevelsatthe12mg/kg/weekdosebyWesternblot,irrespectiveofapo(a)protein

isoformsize,whileinthesalinecohort,nochangesinexpressionlevelswereobservedinanyof

theplasmasamples.TheseresultsdemonstratethatISIS-APO(a)Rxtreatmentishighlyeffective

inloweringplasmaapo(a)levelsinnonhumanprimatesregardlessofindividualvariationin

isoformsorcirculatingconcentrations.

ISIS-APO(a)RxPhase1Trial

Adouble-blinded,placebo-controlled,dose-escalationPhaseItrialinhealthyvolunteers

withLp(a)concentrationof>25nmol/L(>10mg/dL)wasinitiatedtoassesstheefficacyand

safetyandpharmacokineticsofISIS-APO(a)Rx(19).Atotalof16subjectswereenrolledintothe

APO(a)Rxsingle-dosecohortsand31subjectsinthemultiple-dosecohorts.Participantswere

randomlyassignedtoreceiveISIS-APO(a)Rxbysubcutaneousinjection(50mg,100mg,200mg,

or400mg)orplacebo(3:1)inthesingle-dosepartofthestudyortoreceivesixsubcutaneous

injectionsofISIS-APO(a)Rx(100mg,200mg,or300mg,foratotaldoseexposureof600mg,

1200mg,or1800mg)orplacebo(4:1)duringa4weekperiodinthemulti-dosepartofthe

study.

Inthemulti-dosecohort,ISIS-APO(a)Rx(100–300mg)resultedindose-dependent,mean

percentagedecreasesinplasmaLp(a)concentrationof39·6%frombaselineinthe100mg

group(p=0·005),59·0%inthe200mggroup(p=0·001),and77·8%inthe300mggroup

(p=0·001).Thelargestdecreaseinanindividualpatientwas88.8%atday36aftermultiple

dosesof300mgISIS-APO(a)Rx.Maximumplasmaconcentrations(Cmax)ofISIS-APO(a)Rxwere

dose-dependentoverthestudieddoserange,andCmaxwasfollowedbyaninitial,relatively

rapid,distributionphase.Post-distributionplasmaconcentrationsinthe300mgmulti-dose

cohortreachedsteadystatejustbeforeday36,whichcoincidedwiththenadirofLp(a)and

OxPL-apoBandOxPL-apo(a)response(Figure6).Similarreductionswereobservedinthe

amountofoxidizedphospholipidsassociatedwithapolipoproteinB-100(upto90.2%)and

apolipoprotein(a)(upto93.1%),butnosignificantchangeswerenotedinotherlipoproteins

(Figure7).NosignificantchangeswerenotedinOxPLonplasminogenorplasminogenlevels.

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Inthecombinedmulti-dosecohorts,aninversecorrelationwasnotedbetweenthesize

ofthepredominantlyexpressedapo(a)isoformandbaselineplasmaLp(a)andOxPL-apoB

concentrations.However,therewasnosignificantcorrelationbetweenthemajorapo(a)

isoformandthemeanpercentchangefrombaselinetoday36inLp(a)orOxPL-apoB

concentrations),consistentwiththeindependenceofloweringofLp(a)andOxPL-apoBon

isoformsize(Figure8).Inthecombinedsingle-doseandmulti-dosecohortsatalltimepoints,a

strongcorrelationwasnotedbetweenLp(a)concentrationsandOxPL-apoB(r=0·86,p<0·0001

andLp(a)andOxPL-apo(a)(r=0·91,p<0·0001).

Insummary,ISIS-APO(a)Rxresultedinpotent,dose-dependent,selectivereductionsof

plasmaLp(a)andrepresentsapotentialtherapeuticdrugtoreducetheriskofcardiovascular

diseaseandcalcificaorticvalvestenosisinpatientswithelevatedLp(a)concentration.

AlternativeTherapiestoLowerLp(a)

RecentdatahasdemonstratedthatLp(a)canbesignificantlyloweredby20-40%,with

antisenseoligonucleotidestoapoB(35),monoclonalantibodiestoproproteinconvertase

subtilisin/kexintype9(46-48),andcholesterolestertransferproteininhibitors(49,50).

However,inpatientsatorabovethe80thpercentile,correspondingto~50mg/dLplasmaLp(a)

concentrations,muchgreaterreductionthaniscurrentlyachievedwiththeseindirect

therapeuticagentswouldberequiredtosignificantlyreduceCVrisk,whichisthoughttooccur

atlevelswhichexceed25-30mg/dL(8).Therefore,potentandspecificinhibitorsofLp(a)

representanunmetmedicalneedforhighriskpatients.

FutureDirections

FuturestudiesofISIS-APO(a)Rxwillincludegainingmoreexperienceonthesafetyand

efficacyinvariouspopulationswhereitmaybeusedclinically.Forexample,potential

indicationsmayincludepatientswithelevatedLp(a)levelsandotherwisecontrolledrisk

factors,suchaspatientswithrefractoryangina(51,52),recurrentcardiovascularevents

includingpatientsundergoingapheresisforelevatedLp(a)(53-56),youngpatients(i.e.<50-60

yearsold)withCVD(57),calcificaorticvalvestenosis(9),patientswithFamilial

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Hypercholesterolemiaofwhom40-50%haveLp(a)levels>50mg/dL(35),stroke,particularlyin

thepediatricagegroup(58),chronicrenaldiseaseandsecondaryandprimaryprevention.With

thecurrentpotentASOloweringLp(a)levels80-90%,itmaybepossibletolowerLp(a)levelsin

mostpatientstowhatisconsideredleastatherogenic,i.e.<25-30mg/dL,andtotestthe

hypothesisthatloweringLp(a)levelswillleadtoreductioninCVDevents.

AnattractivepopulationtoreduceLp(a)levelsispatientswithpre-existingaorticvalve

stenosis.Theprevalenceofaorticvalvestenosisisincreasingrapidlyduetotheagingofthe

populationandthereisaclinicalneedtoreducetheprogressionofaorticstenosisand

ultimatelytheneedforaorticvalvereplacement.ElevatedLp(a)levelsandtheLPAsnp

s10455872,whichisassociatedwithelevatedLp(a)levels,havebeenrecentlyidentifiedin

epidemiologicandgenome-wideassociationstudiesaspredictorsofaorticvalvestenosis,aortic

valvereplacementandaorticvalvecalcification(6,8).Ourgrouphasrecentlyevaluatedtherole

ofLp(a)andOxPL-apoB,whichreflectsthebiologicalactivityofLp(a),inpredictingtherateof

progressionofpre-existingaorticstenosisintheASTRONOMER(AorticStenosisProgression

Observation:MeasuringEffectsofRosuvastatin)Trial.ElevatedlevelsofbothLp(a)andOxPL-

apoBpredictedaorticstenosisprogression,measuredbytheannualizedincreaseinpeakaortic

jetvelocityinmeterspersecondperyear(m/s/year)byDopplerechocardiography,aswellas

theneedforaorticvalvereplacementandcardiacdeathduring3.5±1.2yearsoffollow-up

wereassociated(9).TherateofprogressionwasfasterinpatientsinthetoptertilesofLp(a)

(peakaorticjetvelocity:+0.26±0.26vs.+0.17±0.21m/s/year;p<0.005)andOxPL-apoB

(+0.26±0.26m/s/yearvs.+0.17±0.21m/s/year;p<0.01)(Figure9).Thesefindingssupport

thehypothesisthatLp(a)mediatesaorticstenosisprogressionthroughitsassociatedOxPLand

providearationaleforrandomizedtrialsofLp(a)-loweringandOxPL-apoB-loweringtherapiesin

aorticstenosis(Figure10).AclinicaltrialcanbeperformedtoassesswhetherloweringLp(a)

mayreduceprogressionofaorticstenosisandtheneedforaorticvalvereplacement.

Finally,theongoingdevelopmentoftri-antennaryN-acetylgalactosamine(GalNAc3)

conjugatesareexpectedtofurtherenhancethepotencyofASOsbyasmuchas10-20foldfor

mRNAtargetsexpressedinhepaticparenchymalcells(59).Thisispossiblebecausethe

physiologicalligandsforGalNAc3uptake,theasialoglycoproteinreceptors-1and-2,are

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abundantlyandexclusivelyexpressedonhepatocytes(60,61).Suchpotencyimprovementmay

ultimatelyleadtoextendeddosingperiods,suchamonthlyorquarterly,orevenorally

bioavailableantisensedrugs,greatlyenhancingtheeaseofadministrationandutilityofthis

emergingtherapeuticplatform.

Acknowledgements

WethankTracyReigleforoptimizationofthefigures.

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FigureLegends

Figure1.MechanismbywhichISIS-APO(a)Rxsuppressesapo(a)proteinsynthesis.Aubiquitous

intracellularribonucleaseRNaseH1recognizestheRNA:DNAduplexformedwhenISIS-APO(a)Rx

bindstothecomplementaryapo(a)mRNAsequenceandcleavesthetarget,therebyreducing

apo(a)proteinandpreventinggenerationofLp(a)particles.TG=triglyceride.PL=phospholipid.

FC=freecholesterol.CE=cholesterylester.KIV=kringleIVrepeats.KV=kringleVrepeat.

OxPL=oxidizedphospholipid.apo(a)=apolipoprotein(a).apoB-100=apolipoproteinB-100.This

figurewasadaptedfromAlbersJJetal(62)andTsimikasetal(19)withpermission.

Figure2.EffectofmipomersenandISIS144367onLp(a)andapo(a)plasmalevels.A-Mean

percentagechangeinapo(B),Lp(a)andtotalapo(a)levelsin8K-apo(a)miceinresponseto

mipomersentargetinghumanapoB.B-Meanpercentagechangeintotalapo(a)levelsin12K-

apo(a)miceinresponsetoISIS-144367.P-valuesrepresentdifferencescomparedtobaseline

values.ReprintedwithpermissionfromMerkietal(30)andMerkietal(12).

Figure3.EffectofAPO(a)Rxonmurineliverapo(a)mRNAexpressionandplasmaapo(a)levels.

Apo(a)livermRNA(panelA)andapo(a)plasmaproteinlevels(panelB)weremeasuredafter2

weeksofISIS-APO(a)Rxadministrationof1.5,5,15and50mg/kg/wk,orsalineinLPAtransgenic

mice(n=4/group).Dosingwasperformedonceweeklybyintraperitonealinjectionfor2weeks.

Mean±standarddeviation(SD)areplottedforeachanalyteasapercentageofthesaline

control.

Figure4.EffectofISIS-APO(a)Rxoncynomolgousmonkeyliverapo(a)mRNAandplasmaLp(a)

levels.A-Cynomolgousmonkeyhepaticapo(a)mRNAsuppressionafter13weekadministration

ofISIS-APO(a)Rxatdosesof4,8,12and40mg/kg/wk.Theheterogeneityofapo(a)mRNAliver

expressionisreadilyapparentwithinthesalineadministeredgroup(n=6).Mean±standard

deviation(SD)areplottedforliverapo(a)mRNAasapercentageofthesalineadministered

control.B-Cynomolgousmonkeyplasmaapo(a)levelsafter13weekadministrationofISIS-

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APO(a)Rxatdosesof0,4,8,12and40mg/kg/wk.Mean±standarddeviation(SD)areplotted

forplasmaapo(a)mRNAasapercentagechangefromDay1baselines.*Statisticallysignificant

differenceusingANOVAanalysisfollowedbyDunnett’smulticomparisonposthoctest(p<0.05).

Figure5.EffectofISIS-APO(a)Rxoncynomolgousmonkeyapo(a)proteinexpressionandplasma

Lp(a)levels.A-Westernanalysisofmaleandfemalecynomolgusmonkeyplasmaapo(a)protein

levelsatDay1predoseandDay93postdoseinsalineand12mg/kg/wkISIS-APO(a)Rx

administeredgroups.Plasmaproteinwasdiluted1:100andseparatedbyPAGEona4-12%Tris

GlycineGel.Apo(a)wasdetectedusingtheLPA4anti-apo(a)antibody.B-Plasmasamplesfor

theindividualmonkeysampleswereanalyzedonaBeckmanclinicalanalyzerusingRandox™

isoform-independentmeasurementsshowninA.Apo(a)levelsareexpressedinmg/dL.

Figure6.PlasmaISIS-APO(a)Rxtroughconcentrationsmeasured7daysafterthelastdoseinthe

300mgdosecohortinrelationtochangeinconcentrationofplasmaLp(a),OxPL-apoB,and

OxPL-apo(a).Theshadedarearepresentsthedosingwindow,andarrowsindicatedosingat

days1,3,5,8,15,and22.Lp(a)=lipoproteina.OxPL-apoB=oxidizedphospholipidon

apolipoproteinB.OxPL-apo(a)=oxidizedphospholipidonapolipoprotein(a).Thisfigurewas

adaptedfromTsimikasetal(19)withpermission.

Figure7.MeanpercentchangeinLp(a),OxPL-apoB,OxPL-apo(a),totalcholesterol,LDL-C,apoB,

HDL-C,andtriglyceridesfrombaselinetoday36amongthedifferentdosesinthemulti-dose

groupsLp(a)=lipoproteina.OxPL-apoB=oxidizedphospholipidonapolipoproteinB.OxPL-

apo(a)=oxidizedphospholipidonapolipoprotein(a).TC=totalcholesterol.LDL-C=low-density

lipoproteinC.ApoB=apolipoproteinB.HDL-C=high-densitylipoproteinC.TG=triglycerides.

ns=non-significant.*p=0.020.†p≤0.008.‡p≤0.001comparedwithplacebo.Thisfigurewas

adaptedfromTsimikasetal(19)withpermission.

Figure8.Mean%changeinLp(a),OxPL-apoBandOxPL-apo(a)atDay36inthe300mg

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multiple-dosegroup,demonstratingthattheextentofloweringwasindependentofbaseline

Lp(a)levels.X-axisshowsindividualpatientsdenotedA-GandtheirbaselineLp(a)levels.This

figurewasadaptedfromTsimikasetal(19)withpermission.

Figure9.CalcificaorticvalvestenosisprogressionrateaccordingtoplasmalevelsofLp(a)and

OxPL-apoB.Annualizedprogressionrateofpeakvelocityacrosstheaorticvalve(Vpeak)is

comparedbytertilesinthewholecohortforLp(a)(A)andforOxPL-apoB(B)andafter

dichotomizationbymedianage(CandD).*p<0.05tertile3(>58.5mg/dl)comparedwith

tertiles1and2(≤58.5mg/dl)ofLp(a)–age≤57group;†p<0.05tertile3(>5.5nM)compared

withtertiles1and2(≤5.5nM)ofOxPL-apoB–age≤57group.Errorbars=SEM.Lp(a)=

lipoprotein(a);OxPL-apoB=oxidizedphospholipidsonapolipoproteinB-100;Vpeak,peakaortic

jetvelocity.ThisfigurewasadaptedfromCapouladeetal(9)withpermission.

Figure10.RelationshipofLp(a)andOxPLwiththedevelopmentandprogressionofcalcific

aorticvalvestenosis.ElevatedLp(a)andOxPLplasmalevelspredictclinicalprogressionofaortic

valvedisease.PatientswithhighLp(a)andOxPLplasmalevelshaveasignificantlyfastercalcific

aorticvalvestenosisprogressionrate,representedbyhigherpeakaorticjetvelocityandlarger

valvularcalcification,comparedwiththosewithlowLp(a)andOxPLplasmalevels.Lp(a)=

lipoprotein(a);OxPL=oxidizedphospholipids.ThisfigurewasadaptedfromCapouladeetal(9)

withpermission.

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Table1.ISIS-APO(a)Rxcomplementarybindingsiteswithinthehumanapo(a)transcript

ISIS-APO(a)Rx

bindingsite

PositiononNM_005577.2

apo(a)mRNAtranscript

Bindingsiteon

1stexon

Bindingsiteon

2ndexon

Exon4:5 505-524bp CTTGTTC TGCTCAGTCGGTG

Exon6:7 847-866bp CTTGTTC TGCTCAGTCGGTG

Exon8:9 1189-1208bp CTTGTTC TGCTCAGTCGGTG

Exon10:11 1531-1550bp CTTGTTC TGCTCAGTCGGTG

Exon12:13 1873-1892bp CTTGTTC TGCTCAGTCGGTG

Exon14:15 2215-2234bp CTTGTTC TGCTCAGTCGGTG

Exon16:17 2557-2576bp CTTGTTC TGCTCAGTCGGTG

Exon18:19 2899-2918bp CTTGTTC TGCTCAGTTGGTG

Exon22:23 3583-3602bp CTTCTTC TGCTCCGTTGGTG

Exon24:25 3901-3920bp CTTGTTC TGCTCCGTTGGTG

Exon28:29 4584-4604bp CTTGTTC TTCTCAGGTGGTG

Exon30:31 4927-4946bp CTGCTTC TGCTCAGTTGGTG

ISIS-APO(a)Rxcomplementarybindingsiteswithinthehumanapo(a)transcript(Genbank

Accession#NM_005577.2atposition3901-3920bp.ISIS-APO(a)Rxwasdesignedtoperfectly

matchonlytheexon24:25splicesite(indicatedwithboldletters)butmayalsobindat11other

apo(a)exonsplicesitescontaining1-4mismatchednucleotides(indicatedbygrayletters).

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Table2.EffectofISIS-APO(a)Rxonapo(a)andplasminogenmRNAincynomolgusmonkeys

Treatment Dose DurationApo(a)mRNA

(%Saline)

Plasminogen

mRNA

(%Saline)

TC HDL-C LDL-C apoB

Saline 0mg/kg/wk 13weeks 100±26 100±3 89±7 73±8 103±8 123±12

APO(a)Rx 40mg/kg/wk 13weeks 10±3* 107±3 96±6 83±6 109±9 122±18

ISIS-APO(a)Rxwereadministeredtoleancynomolgusmonkeysat40mg/kg/wkover13weeks

(n=4/group).Aloadingregimenof3dosesinfirstweekfollowedbyonceperweek

subcutaneousadministrationwasutilized.Dataareexpressedasthemeanpercentageof

valuesobservedinsaline(±SEM)treatedmonkeysforapo(a)andplasminogenmRNAlevels.

Plasmatotalcholesterol(TC),HDL-C,LDL-CandapoBproteinareexpressedasapercentageof

baselinelevels.*IndicatesstatisticallysignificantfromsalineusingMannWhitneytwo-tailed

test(p<0.05).

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Figure1.ProposedmechanismbywhichISIS-APO(a)Rxsuppressesapo(a)proteintranslation

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Figure2.EffectofmipomersenandISIS144367onLp(a)andapo(a)plasmalevels.

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Figure3.EffectofISIS-APO(a)Rxonliverapo(a)mRNAandapo(a)proteinlevelsinLPA

transgenicmouseprimaryhepatocytes.

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Figure4.EffectofISIS-APO(a)Rxoncynomolgousmonkeyliverapo(a)mRNAandplasmaapo(a)

levels.

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Figure5.EffectofISIS-APO(a)Rxoncynomolgousmonkeyapo(a)proteinexpressionandplasma

apo(a)levels.

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Figure6.PlasmaISIS-APO(a)Rxtroughconcentrationsinthe300mgdosecohortinrelationto

changeinconcentrationofplasmaLp(a),OxPL-apoB,andOxPL-apo(a).

Lp(a), % changeOxPL-‐apoB, % change

ISIS-‐APO(a)Rx Plasma conc, ng/ml

OxPL-‐apo(a), % change

Mea

n %

cha

nge

from

bas

elin

e

-100

-80

-60

-40

-20

0

20

40

60

80

0 20 40 60 80 100 120Study Day

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Figure7.ChangeinLp(a),OxPL-apoB,OxPL-apo(a),totalcholesterol,LDL-C,apoB,HDL-C,and

triglyceridesfrombaselinetoday36amongthedifferentdosesinthemulti-dosegroups.

-90

-70

-50

-30

-10

10

30

50

Lp(a) OxPL-apoB OxPL-apo(a) TC LDL-C ApoB HDL-C TG

Mea

n %

cha

nge

from

bas

elin

e to

Day

36

PlaceboISIS-APO(a)Rx 100 mg

ISIS-APO(a)Rx 200 mgISIS-APO(a)Rx 300 mg

**

***

***

***

* **

P = NS

*

***

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Figure8.Mean%changeinLp(a),OxPL-apoBandOxPL-apo(a)atDay36inthe300mg

multiple-dosegroup.

-100

-80

-60

-40

-20

0

20

40

A-23.9 B-36.2 C-75.3 D-128.9 E-148.2 F-194.6 G-220.1

% c

hang

e fro

m b

asel

ine

to D

ay 3

6

Baseline Lp(a) levels in individual patients, nmol/L

% change Lp(a)

% Change OxPL-apoB

% change OxPL-apo(a)

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Figure9.CalcificaorticvalvestenosisprogressionrateaccordingtoplasmalevelsofLp(a)and

OxPL-apoB.

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Figure10.RelationshipofLp(a)andOxPLwiththedevelopmentandprogressionofcalcific

aorticvalvestenosis.

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Journal of Lipid Research Volume 57, 2016 2235

The authors of “Antisense inhibition of apolipoprotein(a) to lower plasma lipoprotein(a) levels in humans” (J. Lipid Res. 2016. 57: 340–351) have advised the Journal that there was an error in the legend to Table 1. The corrected table legend should read “ISIS-APO(a)Rx complementary binding sites within the human apo(a) transcript (GenBank acces-sion NM_005577.2) at position 3901-3920. ISIS-APO(a)Rx was designed to perfectly match only the exon 24-25 splice site (indicated with bold type) but may also bind at 11 other apo(a) exon splice sites containing one to three mismatched nucleotides (indicated by underlined letters).” Additionally, on page 343 under the “Identification of a Second Genera-tion Antisense Drug to Human apo(a)” section, “ISIS_APO(a)Rx also has the potential to bind to 11 alternative sites within the transcript containing one to four mismatched nucleotides” should read “ISIS_APO(a)Rx also has the potential to bind to 11 alternative sites within the transcript containing one to three mismatched nucleotides.”

DOI 10.1194/jlr.R052258ERR

ERRATUM