Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B

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Title:
Role of the vector genome and underlying factor IX mutation in immune responses to AAV gene therapy for hemophilia B
Series Title:
Journal of Translational Medicine
Physical Description:
Mixed Material
Creator:
Geoffrey L Rogers
Ashley T Martino
Irene Zolotukhin
Hildegund CJ Ertl
Roland W Herzog
Publisher:
Journal of Translational Medicine

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Subjects / Keywords:
AAV
Gene therapy
Hemophilia B
Factor IX,
Immune response

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Abstract:
Background: Self-complementary adeno-associated virus (scAAV) vectors have become a desirable vector for therapeutic gene transfer due to their ability to produce greater levels of transgene than single-stranded AAV (ssAAV). However, recent reports have suggested that scAAV vectors are more immunogenic than ssAAV. In this study, we investigated the effects of a self-complementary genome during gene therapy with a therapeutic protein, human factor IX (hF.IX). Methods: Hemophilia B mice were injected intramuscularly with ss or scAAV1 vectors expressing hF.IX. The outcome of gene transfer was assessed, including transgene expression as well as antibody and CD8+ T cell responses to hF.IX. Results: Self-complementary AAV1 vectors induced similar antibody responses (which eliminated systemic hF.IX expression) but stronger CD8+ T cell responses to hF.IX relative to ssAAV1 in mice with F9 gene deletion. As a result, hF.IX-expressing muscle fibers were effectively eliminated in scAAV-treated mice. In contrast, mice with F9 nonsense mutation (late stop codon) lacked antibody or T cell responses, thus showing long-term expression regardless of the vector genome. Conclusions: The nature of the AAV genome can impact the CD8+ T cell response to the therapeutic transgene product. In mice with endogenous hF.IX expression, however, this enhanced immunogenicity did not break tolerance to hF.IX, suggesting that the underlying mutation is a more important risk factor for transgene-specific immunity than the molecular form of the AAV genome.

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University of Florida
Holding Location:
University of Florida
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All rights reserved by the source institution.
System ID:
AA00020086:00001

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RESEARCHOpenAccessRoleofthevectorgenomeandunderlyingfactor IXmutationinimmuneresponsestoAAVgene therapyforhemophiliaBGeoffreyLRogers1,AshleyTMartino1,IreneZolotukhin1,HildegundCJErtl2andRolandWHerzog1*AbstractBackground: Self-complementaryadeno-associatedvirus(scAAV)vectorshavebecomeadesirablevectorfor therapeuticgenetransferduetotheirabilitytoproducegreaterlevelsoftransgenethansingle-strandedAAV (ssAAV).However,recentreportshavesuggestedthatscAAVvectorsaremoreimmunogenicthanssAAV.Inthis study,weinvestigatedtheeffectsofaself-complementarygenomeduringgenetherapywithatherapeuticprotein, humanfactorIX(hF.IX). Methods: HemophiliaBmicewereinjectedintramuscularlywithssorscAAV1vectorsexpressinghF.IX.The outcomeofgenetransferwasassessed,includingtransgeneexpressionaswellasantibodyandCD8+Tcell responsestohF.IX. Results: Self-complementaryAAV1vectorsinducedsimilarantibodyresponses(whicheliminatedsystemichF.IX expression)butstrongerCD8+TcellresponsestohF.IXrelativetossAAV1inmicewith F9 genedeletion.Asa result,hF.IX-expressingmusclefiberswereeffectivelyeliminatedinscAAV-treatedmice.Incontrast,micewith F9 nonsensemutation(latestopcodon)lackedantibodyorTcellresponses,thusshowinglong-termexpression regardlessofthevectorgenome. Conclusions: ThenatureoftheAAVgenomecanimpacttheCD8+Tcellresponsetothetherapeutictransgene product.InmicewithendogenoushF.IXexpression,however,thisenhancedimmunogenicitydidnotbreak tolerancetohF.IX,suggestingthattheunderlyingmutationisamoreimportantriskfactorfortransgene-specific immunitythanthemolecularformoftheAAVgenome. Keywords: AAV,Genetherapy,HemophiliaB,FactorIX,ImmuneresponseBackgroundHemophiliaBistheX-linkedmonogeneticdisorder causedbythelossoffunctionalcoagulationfactorIX (F.IX),resultinginadeficiencyintheabilityofbloodto clot.Inadditiontoincreasedpropensityforbleeding aftertraumaorinjury,spontaneousbleedscanoccurin capillaries,particularlyinthejoints,resultingintissue damageovertime.Bleedsintocriticalclosedspacescan belife-threatening.Currently,hemophiliaBistreated byintravenousadministrationofF.IXconcentrate, eitherplasma-derivedorrecombinant,inorderto restorehemostasis.Becauseoftheshorthalf-lifeofthe proteinincirculation,frequentinjectionsarerequired toprovideprophylaxisortotreatpatientswithsevere diseaseondemand.Genetherapyrepresentsanattractivealternativetoproteinreplacementtherapy,asit wouldinvolveasingleinjectiontoprovidelong-termintrinsicproductionofF.IX. AmongpotentialgenetherapiesforhemophiliaB,the useofadeno-associatedvirus(AAV)asagenedelivery vectorhasshownthemostsuccesstodate[1].AAVisa dependovirus,aparvovirusthatisunabletoreplicatein theabsenceofahelpervirus(typicallyadenovirus).For useasagenetherapyvector,allviralgenesareremoved, leavingonlytheinvertedterminalrepeatsrequiredfor packagingaroundthetransgenicconstruct.Thevarious *Correspondence: rherzog@ufl.edu1DepartmentofPediatrics,DivisionofCellularandMolecularTherapy, UniversityofFlorida,Gainesville,Florida,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Rogersetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.TheCreativeCommonsPublicDomainDedication waiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwise stated.Rogers etal.JournalofTranslationalMedicine 2014, 12 :25 http://www.translational-medicine.com/content/12/1/25

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serotypesofAAVhavedifferenttropisms,whichallow forgenetransfertonumeroustargettissues[2].Forinstance,AAV1caneffectivelytransduceskeletalmuscle, whileAAV8hasstrongtropismforlivertissue.Preclinicalstudiesinanimalsestablishedthattheriskof immuneresponsestoF.IXissubstantiallyaffectedby therouteofvectoradministrationandbytheunderlying geneticdefect. F9 nullmutations(completeabsenceof protein,forexampleresultingfromagenedeletion)are mostlikelyassociatedwithstrongimmuneresponse, whilemutationspreservingsomelevelofendogenous, albeitnon-functionalF.IXexpression,reducetherisk forimmuneresponses[3-6]. Recentclinicaltrialsarebasedonliver-directedgene transfer.HepatocytesarethenormalsiteofF.IXsynthesis.Furthermore,highlevelsofantigenexpressionin hepatocytespromoteinductionofregulatoryTcells, resultinginimmunetoleranceinductiontothetransgeneproduct.Thisapproachisevenabletoreversean ongoingantibodyresponseagainstF.IX[4,7,8].Sustained expressionofF.IXbyhepaticgenetransferhasnowbeen demonstratedinhemophiliaBpatients,followingsuccessesinlargeanimalsmodel,includingnon-human primatesandhemophiliaBdogs[9-11]. AAVvectorstraditionallycontainasingle-stranded DNAgenome(ssAAV)withapackaginglimitofapproximately5kb.Bymodifyingoneoftheinverted terminalrepeats,itispossibletoforcethevirustopackageaself-complementarydouble-strandedDNAgenome(scAAV),therebybypassingtheneedtofor second-strandsynthesis,oneoftherate-limitingstepsin AAVtransduction[12].Adisadvantageofthisstrategyis thefurtherreducedpackaginglimit.Nonetheless,scAAV vectorsexpressingF.IXfromliver-specificpromoters havebeenoptimizedandarecurrentlyusedinclinical trials[9].Inadditiontomorerapidtransgeneexpression,scAAVvectorsoftenproducehighertransgene levelsthanssAAVwithanequivalentinputdose[11].At thesametime,wefoundthatscAAVvectorselicited strongerinnateimmuneresponsesintheliverthan ssAAV,likelybecauseofenhancedtoll-likereceptor 9(TLR9)signaling.Consistentwithpriorstudiesby others,hepaticinnateimmuneresponsestoAAVvectors weredependentonTLR9,anendosomalreceptorthat recognizesunmethylatedCpGDNAmotifs[13-15].In ourhepaticgenetransfermodel,theheightenedinnate responsedidnotincreaseadaptiveimmuneresponsesto theF.IXtransgeneproductbutcausedmodestincreases inBandTcellresponsestothecapsidantigensofthe vector. Skeletalmusclerepresentsanalternativetargettissue forAAV-F.IXgenetransfer.Upongenetransfermyofibersarecapableofproducingbiologicallyactivematerial,andthefirstclinicaltrialonAAV-F.IXgenetransfer utilizedintramuscularinjectionsatmultipleskeletal musclesitesastherouteofvectoradministration [16-19].F.IX-expressingmusclefibersmaypersistin humansforatleast10yearsafterinitialgenetransfer [20].However,aconcernaboutmuscle-directedgene transferistheincreasedriskofimmuneresponses againstF.IX.Hence,inthisstudywechosethemoreimmunogenicintramuscularroutetoassessthepotential forBandTcellresponsesagainstF.IXasafunctionof thevectorgenome(scAAVvsssAAV)andtheunderlying F9 genemutation.Theresultsshowastrongerand moredestructiveCD8+TcellresponseusingscAAVin micewitha F9 genedeletion,whilemiceexpressing truncatedhF.IXremainedtoleranttoF.IXregardlessof vectorgenomeconformation.MethodsAnimalstrainsandexperimentsHemophiliaBmicewithtargeteddeletionofmurine F9 ( ‘ HB ’ )hadbeenbredonC3H/HeJbackgroundfor>10 generations[21].MicetransgenicfortruncatedhF.IX (human F9 complementaryDNAincludinga0.3-kbportionofintronIexpressedfromliver specifictransthyretinpromoter)wereaspublished[22].Theseanimals expresshF.IXwithlatestopcodonataminoacidresidue 338( ‘ LS ’ ).ThislinewasoriginallynumberedasLS-37 andcontains6copiesofthehF.IXgene[22].Theline wasrepeatedlybackcrossedontoC3H/HeJbackground (>10generations),andfinallycrossedwithHBmicein ordertoeliminateendogenousmurineF.IXexpression [3].Animalswerehousedunderspecificpathogen-free conditionsattheUniversityofFloridaandtreatedunder InstitutionalAnimalCareandUseCommittee-approved protocols.Allanimalsweremaleand6 … 8weeksoldat theonsetoftheexperiments;allcohortscontainedat least4micepergroup. AAVvectorswereadministeredintramuscularlyinto twosites:quadricepsandtibialisanteriorofonehindlimb, aspreviouslydescribed[23].Plasmasampleswerecollectedbytailbleedintocitratebufferasdescribed[21].AAVvectorsssAAVvectorexpressinghumanF.IXcDNA(including a1.4-kbportionofintronI)fromtheCMVIEenhancer/promoterwasaspublished[19].Forconstruction ofscAAV,thehumanF.IXcodingsequence(lacking intronicor3 untranslatedsequences)wasclonedinto anscAAV-CMV-GFPconstruct,replacingtheGFPsequence.Thisconstructcontainsasmall -globin/IgG chimericintron.Vectorgenomeswerepackagedinto AAVserotype1capsidbytripletransfectionofHEK-293 cells.Vectorparticleswerepurifiedbyiodixanolgradient centrifugation,andvectortitersdeterminedbydotblotRogers etal.JournalofTranslationalMedicine 2014, 12 :25Page2of10 http://www.translational-medicine.com/content/12/1/25

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hybridizationandconfirmedbyWesternblotusinga referencestandardofknowntiterforcomparison.AnalysisofplasmasamplesPlasmawasanalyzedforhF.IXexpression,anti-hF.IX IgG1,andanti-AAV1IgG2abyenzyme-linkedimmunosorbentassay(ELISA)aspreviouslydescribed[13,21]. Fortheanti-capsidantibodyELISAs,samplewellswere coatedwith2.5109vg/wellintactAAV1particles.The assayforanti-hF.IXIgG1wassensitiveto~200ng/mL. Anti-hF.IXinhibitoryactivitywasassessedusingthe Bethesdaassay,aspreviouslydescribed[3].OneBethesda unit(BU)representstheinhibitionof50%ofclotting activity.ClottingassayswereperformedonaSTart HemostasisAnalyzer(DiagnosticaStago,Parsippany,NJ).ELISPOTassaysEnzyme-linkedimmunosorbentspot(ELISPOT)assays wereperformedtoenumeratehF.IX-specificCD8+T cellsinmousespleens,aspreviouslydescribed[3,24]. Briefly,splenocyteswereplatedat1106cells/well,and stimulatedwithmediaalone,staphylococcalenterotoxin B(ToxinTechnologies,Sarasota,FL;1ug/mL),orthe immunodominantCD8epitopeofhF.IXfortheC3HHeJbackground(p74,Anaspec,SanJose,CA;10ug/mL) [3].Analyseswereperformedintriplicateonindividualmice.Afterstimulationfor20hours,plateswere harvestedandIFNspot-formingunits(SFU)were detectedandcountedusingtheImmunoSpotAnalyzer (CellularTechnology,ShakerHeights,OH).Resultswere calculatedasspot-formingunitsper106totalcells.ImmunohistochemistryImmunohistochemistrywasperformedusingfluorescent antibodiesonfrozenandcryosectionedtissue,aspreviouslydescribed[25].Briefly,muscletissuewasharvestedandfrozeninliquidN2-cooled2-methylbutane. Cryosections(10 m)oftissuewerefixedinacetone atroomtemperature,blockedwith5%donkeyserum (Sigma,St.Louis,MO),andstainedwithratanti-CD8 (eBioscience,SanDiego,CA)andgoatanti-hF.IX (AffinityBiologicals,Ontario,Canada).Secondaryantibodydonkeyanti-ratAlexaFluor488anddonkeyantigoatAlexaFluor568(LifeTechnologies,Eugene,OR) wereusedfordetection.Fluorescencemicroscopywas performedwithaNikonE800microscope(Nikon, Tokyo,Japan).StatisticsResultsarereportedasmeansSEM.Significantdifferencesbetweengroupsweredeterminedwithunpaired Student ’ s t -test. P valuesof<0.05wereconsideredsignificant.AnalyseswereperformedusingGraphPad Prism(SanDiego,CA).ResultsThevectorgenomeaffectstheCD8+Tcellresponseto F.IXinnullmutationmiceToassesstheeffectofascAAVgenomeontheimmune responsetoF.IX,weinjectedhemophiliaB(HB)C3H/HeJ miceintramuscularly(i.m.)with1011vectorgenomes(vg) ofssorscAAVserotype1vectorsexpressinghumanF.IX (hF.IX)underthecontrolofacytomegaloviruspromoter (AAV1-CMV-hF.IX).TheseHBmicehaveatargeteddeletionofthemurine F9 geneandthereforelacktoleranceto F.IXantigen.Inpreviousstudies,wefoundthatssAAV2CMV-hF.IX(serotype2vector)inducedneutralizingantibodyandCD8+TcellresponsesagainsthF.IXuponi.m. injectioninthisstrain[3].Here,weusedserotype1vector,becauseitissuperiorformusclegenetransferand ishenceinclinicaltrial/useformusclegenetransferfor 1-antitrypsindeficiencyandforlipoproteinlipasedeficiency[26-29]. Plasmawasthencollected1,2,and4weekspostinjectiontoassesscirculatingexpressionofhF.IXaswell asantibodyresponsestothetransgeneproduct.One weekaftervectorinjection,expressionofhF.IXwasdetectedinmicethatreceivedssorscAAV1(Figure1A). Attwoweeksandthereafter,though,circulatinghF.IX wasnotdetectedineithergroupofanimals. CorrespondingwiththelossofhF.IXexpressionin plasma,antibodiesagainsthF.IXwerefirstdetected2 weekspost-injectionbyELISA(Figure1B).Consistent withpriorfindings,thesewereoftheIgG1subclass, whereaslevelsofIgG2aandI gG2bwerecomparatively verylowornonexistent(datanotshown)[3,30,31]. Averageanti-hF.IXtiterswerenearlyidenticalforboth ssandscAAVvectors.Toassessthefunctionalityofthis humoralimmuneresponse,weperformedtheBethesda assay,whichmeasurestheabilityofhF.IX-specificantibodies(inhibitors)topreventplasmaclottingactivity. Inhibitortiterslaggedbehindthedetectionofanti-hF. IXIgG1,withnolittleornoinhibitionofclotting detectedaftertwoweeks(Figure1C).After4weeks, averagetitersof~20BUweremeasuredregardless whethermicereceivedssorscAAV1. Twoandfourweekspost-injection,splenocyteswere harvestedtomeasuretheCD8+TcellresponsetohF.IX byELISPOT.Bothvectorsinducedameasurableantigenspecificresponse.However,micethatreceivedscAAV1 hadasignificantlyhighernumberofIFNspot-forming units(SFU)whenstimulatedwiththeimmunodominant CD8epitopeofhF.IXat2weeks(Figure1D).Fourweeks post-injection,allanimalsstillshowedaresponse,which wassimilarforssandscAAV1-treatedmiceatthislater timepoint(Figure1E).BackgroundSFU(mediaandSEB treatments)werehigherat2weeks,possiblyduetoelevatedimmuneactivityatthistimepoint.Inordertoassess whetheractivatedhF.IX-specificCTLsinfiltratedtheRogers etal.JournalofTranslationalMedicine 2014, 12 :25Page3of10 http://www.translational-medicine.com/content/12/1/25

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transducedtissue,immunohistochemicalanalysesof injectedmuscleswereperformed.Twoweekspostinjection,micethatreceivedeitherssorscAAV1had significantCD8+Tcellinfiltration,thoughtherewas moreevidenceoflocalhF.IXproductioninssAAV1treatedmice(Figure2A-B,E-F).Atfourweekspostinjection,muscletransducedwithssAAV1maintained hF.IXexpressionconcomitantwithcontinuedCD8+T cellinfiltrates,whereasmicethatreceivedscAAV1had veryfewtransducedskeletalmusclecellsremaining, andCD8+Tcellinfiltrationhadsubsided(Figure2C-D, G-H).Micewithanonsensemutationfailtomountanimmune responseagainstF.IXregardlessoftheAAVgenomeWiththeindicationthatscAAVvectorsmayinducea strongerCD8+TcellresponsetohF.IX,wenextsought todeterminewhethertheycouldinducearesponsein hemophilicmicewithamutationthatresultsinnonfunctionalhF.IXexpression.Wehadpreviouslyestablishedhemophilicmicecarrying F9 missensemutations oranonsensemutation.Wheninjectedi.m.withAAV2CMV-hF.IXvector,noneofthemiceofeitherofthese linesshowedaCD8+TcellresponsetoF.IX;however, micewithalatestopcodonmutation(ataminoacid residue338ofF.IX,  LS Ž line)producedantibodies againsthF.IX,indicatingthatthesemicewerenotfully toleranttohF.IX[3].Thus,wechosetheLSlineof hemophilicmicetotestwhetheri.m.administrationof anscAAV1vectorcouldbreakCD8+Tcelltoleranceto hF.IX. OneweekaftergenetransferwitheitherscorssAAV1 vectors,circulatinghF.IXwasdetectedatlevelssimilar tothosereportedaboveforHBnullmutationmice.At2 and4weekspost-injection,hF.IXexpressionincreased andpersisted,withexpressionlevelsinssAAV1-treated miceabout3-foldhigherthanscAAV1-injectedmice after4weeks(Figure3A).NoneoftheLSmicedevelopedantibodies/inhibitorsagainsthF.IXoverthecourse oftheexperiment(Figure3B-C).After4weeks,splenocyteswereonceagainharvestedtomeasuretheCD8+TcellresponsestohF.IXbyELISPOT.Aswiththe 01234 0 50 100 150 200 ssAAV1 scAAV1 nsWeekshF.IX (ng/mL) 01234 0 10 20 30 ssAAV1 scAAV1 WeeksBethesda Units (BU) 01234 0 5000 10000 15000 ssAAV1 scAAV1 Weeksanti-hF.IX IgG1 (ng/mL) 4 weeks ssAAV1scAAV1 0 200 400 600Media Peptide SEB ****nsIFNSFU/106 cells A B C 2 weeks ssAAV1scAAV1 0 200 400 600**Media Peptide SEB ** ***IFNSFU/106 cells D E Figure1 OutcomeofgenetransferwithssorscAAV1inHBmice. HBmicewereinjectedi.m.with1011vgofssorscAAV1-CMV-hF.IX ( n =4/group).Plasmawascollected1,2,and4weekspost-injection. (A) CirculatinghF.IXlevelsweremeasuredbyELISA. (B) Anti-hF.IXIgG1 levelsinplasmaweremeasuredbyELISA. (C) Bethesdatiter.OneBUrepresentstheinhibitionof50%ofclottingactivity. (D-E) Splenocyteswere harvestedandrestimulatedwithmediaalone,theCD8epitopeofhF.IX,orSEB,andIFNspot-formingunits(per106cells)weremeasuredby ELISPOT.Measurementswereperformedonindividualanimalstwoweeks (D) orfourweeks (E) post-injection.DatapointsareaveragesSEM. Resultsarerepresentativeofatleasttwoindependentexperiments. *P<0.05,**P<0.01,***P<0.001,ns=notsignificant Rogers etal.JournalofTranslationalMedicine 2014, 12 :25Page4of10 http://www.translational-medicine.com/content/12/1/25

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humoralimmuneresponse,therewasnoevidenceof splenichF.IX-specificCD8+TcellsinLSmicetreated witheithervector(Figure3D).Thesituationwithinthe muscleitselfreflectedwhathadbeenobservedsystemically.MiceinjectedwitheitherssorscAAV1showed similartransductionofskeletalmusclewithoutevidence ofinfiltratingCD8+Tcells(Figure4).Insummary,use ofscAAVvectordidnotincreasetheriskforhumoralor cellularimmuneresponsestothehF.IXtransgeneproductinthecontextoftheLSnonsensemutation. SinceLSmicedisplayedhigherhF.IXexpressionlevels fromssAAV1vectorscomparedtoscAAV1inthe absenceofanimmuneresponse,wewantedtoverifythe functionalityoftheself-complementaryvectoronanotherbackground.Thus,RAG-deficientC57BL/6mice thatlackBandTcellswereinjectedintramuscularly with1011vgofeithervector.Inthesemice,circulating hF.IXlevelsweresignificantlyhigherinanimalstreated withscAAV1,suggestingthattheinversioninexpression levelsobservedintheLSmicemaybeastrain-specific effect(Figure3E).Anti-capsidantibodiesarenotalteredbyscAAVvectorsFinally,weinvestigatedwhetherthevectorgenomemay alterantibodyresponsesagainstAAVcapsid.Fourweeks afteri.m.injectionofssorscAAV1,wemeasuredthe formationofAAV1-specificantibodies(whicharetypicallyofaTh1associatedsubclasssuchasIgG2a)in plasmabyELISA[13,32].Atthistimepoint,levelsof anti-AAV1IgG2awerecomparablewhethermicereceivedssorscAAV1(Figure5).Aswiththetransgene, capsid-specificantibodyformationwasnotenhancedby scAAVvectorsrelativetossAAV.DiscussionAmajorconcerningenereplacementtherapyisthepotentialforadaptiveimmuneresponsestothetherapeutic transgeneproduct,whichmayberecognizedbythe Figure2 LocalhF.IXexpressionandCD8infiltrationinHBmice. SkeletalmusclefromHBmiceinjectedi.m.with1011vgssorscAAV1 ( n =4/group)washarvested,cryosectioned,andstainedforhF.IX(red)andCD8(green).NucleiwerevisualizedwithDAPI(blue).Twoweeks post-injection,tissuewasanalyzedfrommiceinjectedwithssAAV1 (A-B) orscAAV1 (E-F) .Afterfourweeks,skeletalmusclewasstainedfrommice injectedwithssAAV1 (C-D) orscAAV1 (G-H) .Representativeimagesfromtwomiceareshownforeachcondition.Thescalebarrepresents 100 m.Resultsarerepresentativeofatleasttwoindependentexperiments. Rogers etal.JournalofTranslationalMedicine 2014, 12 :25Page5of10 http://www.translational-medicine.com/content/12/1/25

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01234 0 50 100 150 200 ssAAV1 scAAV1 *** *** ***WeekshF.IX (ng/mL) 01234 0 10 20 30 ssAAV1 scAAV1 WeeksBethesda Units (BU) 01234 0 5000 10000 15000 ssAAV1 scAAV1 Weeksanti-hF.IX IgG1 (ng/mL)AB CD ssAAV1scAAV1 0 200 400 600Media Peptide SEB IF NSFU/106 cellsE ssAAV1scAAV1 0 200 400 600 800 *hF.IX (ng/mL) Figure3 OutcomeofgenetransferwithssorscAAV1inLSmice. LSmicewereinjectedi.m.with1011vgofssorscAAV1-CMV-hF.IX ( n =4/group).Plasmawascollected1,2,and4weekspost-injection. (A) CirculatinghF.IXlevelsweremeasuredbyELISA. (B) Anti-hF.IXIgG1levelsin plasmaweremeasuredbyELISA. (C) Bethesdatiter.OneBUrepresentstheinhibitionof50%ofclottingactivity. (D) Splenocyteswereharvestedfour weekspost-injectionandrestimulatedwithmediaalone,theCD8epitopeofhF.IX,orSEB,andIFNspot-formingunits(per106cells)weremeasured byELISPOT.Measurementswereperformedonindividualanimals. (E) CirculatinghF.IXlevelsinC57BL/6RAG / mice2weekspost-injectionwithssor scAAV1-CMV-hF.IX( n =4/group).DatapointsareaveragesSEM.Resultsarerepresentativeofatleasttwoindependentexperiments. *P<0.05, ***P<0.001,ns=notsignificant Figure4 LocalhF.IXexpressionandCD8infiltrationinLSmice. SkeletalmusclefromLSmiceinjectedi.m.with1011vgssorscAAV1 ( n =4/group)washarvested,cryosectioned,andstainedforhF.IX(red )andCD8(green).NucleiwerevisualizedwithDAPI(blue).Fourweeks post-injection,tissuewasharvestedfrommiceinjectedwithssAAV1 (A-B) orscAAV1 (C-D) .Representativeimagesfromtwomiceareshown foreachcondition.Thescalebarrepresents100 m.Resultsarerepresentativeofatleasttwoindependentexperiments. Rogers etal.JournalofTranslationalMedicine 2014, 12 :25Page6of10 http://www.translational-medicine.com/content/12/1/25

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immunesystemasaforeignantigen.OurpreviousstudieswithhemophilicmiceanddogshaveclearlydocumentedamajorrolefortheunderlyingF.IXmutation ontheriskofBandTcellresponsestothetransgene productingenetherapyforhemophiliaB[3,17,23,33]. However,immuneresponsesrequireactivationsignals, whichmaybederivedfrominnateimmunerecognition ofthevector.Hence,thereareanumberofadditional factorsthatinfluencethelikelihood,strength,andcharacteristicsofanimmuneresponse.Amongothers,these includethechoiceanddesignofthevector,dose,and routeofadministration[4,21,34-38].Self-complementaryvectorsmayincreaseimmune responsestothetransgeneproductdependingonthe routeofvectoradministrationSelf-complementaryAAVvectorshavebeenoptimized forF.IXgeneexpressionandhavegatheredgrowing enthusiasmbecauseofthepotentialforimprovedgene transferandexpression[11,39,40].Atthesametime, usingscAAVinsteadofssAAVmaychangeinnateimmunityaswellasthekineticsandmagnitudeoftransgeneexpression.Here,weaddresshowthischangein vectorgenomeconformationmayinfluenceimmune responsestoF.IXduringmuscle-directedgenetransfer. InnateimmuneresponsestoAAVvectorsaretypically weakandtransient,resultinginlimitedinflammatory signals[13,41,42].Nonetheless,wepreviouslyfoundthat scAAVenhancedTLR9-dependentinnateimmuneresponses,resultinginstrongerNFBdependentinflammationoftissueandexpressionofIFNI[13,43].This increasedimmunogenicity,however,didnotaffectF.IXspecificimmuneresponsesandonlymodestlyincreased antibodyformationagainstthevectorinliver-directed genetransfer[13].Hepatictransgeneexpressionoccurs inanenvironmentcharacterizedbyactivedownregulationofimmuneresponses,therebyfavoringinductionofregulatoryTcellsandestablishmentofimmunetolerance[8,44-49]. Ontheotherhand,expressionofawell-characterized vaccineantigen(HIVgag)inskeletalmuscleyieldedstrongerandmorefunctionalCD8+Tcellresponses,which wascharacterizedbygreaterexpressionofcytokinesand effectormarkersaswellasincreasedlyticcapability invivo .Additionally,strongerantibodyresponseswere observedwhenusingscAAVcomparedtossAAVvectors [50].InhemophiliaBmicewitha F9 genedeletion,we reconstitutedsomeofthesefindings:theCD8+TcellresponsesagainsthF.IXwasmorerobustandalsomore functionalusingthescAAVvector,withinfiltratingTcells rapidlyeliminatinghF.IXexpressingmusclefibers.Inthe contextofssAAVgenetransfer,theensuingCD8+Tcell responseresultsinchronicinfiltrationoftransduced musclewithouteliminationofexpression.TheseobservationsareconsistentwithoutpreviousfindingswithssAAV vectors[6].CD8+TcellsinducedbyssAAVhavereduced cytotoxicandproliferativecapacitythatcannotberescued bysecondaryimmunization,mostlikelyduetoTcellexhaustionandapoptosis[50-52].Additionally,ithasbeen suggestedthatregulatoryTcellsinducedbypersistent AAVcapsidsinskeletalmusclewereabletopreventeliminationoftransducedmyocytesbychronicallyinfiltratingCTLsinaclinicaltrialfor 1-antitrypsindeficiency [27].ItisthereforepossiblethatregulatoryTcellscould alsobeinvolvedinourmodel.Althoughnotaddressed here,wepreviouslyfoundthatadministrationofscAAV alsoincreasesCD8+Tcellresponsestocapsidcompared tossAAV[13]. Incontrast,antibodyresponsesagainstvectorortransgeneproductseemlessconsistentlyaffectedbyuseof scAAVgenomes.ThismaybeexplainedbyagreaterdependenceofCD8+TcellresponsesthanofantibodyresponsesonTLR9activationbyAAVvectors[47,53]. InnateimmunesensingofAAVvectorsdependson TLR9andisincreasedwithscAAVduetoincreased TLR9signalingfromthesevectors[13,15].Interestingly, removalofCpGmotifsfromAAVvectorgenomessubstantiallyreducesCD8+Tcellactivationbuthaslittle effectonantibodyformation[47].Ourresultsconcur withthesefindings,asantibodyresponsestobothtransgeneandcapsidwerenotelevatedwithscAAVvectors.Theunderlyingmutationisagreaterdeterminantofthe riskofimmuneresponsestoF.IXthanthevectorgenome conformationPreviously,webredhemophiliaBmiceontotheC3H/HeJ background,whichgiveshigherantibody/inhibitorand CD8+TcellresponsestohF.IXthanothercommonbackgrounds.Micewithanullmutation( F9 genedeletion) showedsuchresponsestohF.IXinmusclegenetransfer ssAAV1scAAV1 0 200 400 600 800 anti-AAV1 IgG2a (ng/mL) ns Figure5 Anti-capsidantibodyresponse. PlasmafromHBorLS miceinjectedi.m.with1011vgofss( n =19)orscAAV1( n =18)was analyzedfortheformationofanti-AAV1IgG2abyELISA4weeks post-injection.Datapointsrepresentindividualmice,andtheerror barsshowmeanSEM. ns=notsignificant. Rogers etal.JournalofTranslationalMedicine 2014, 12 :25Page7of10 http://www.translational-medicine.com/content/12/1/25

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andsuboptimalhepaticgenetransfer[3,30,31,54].These micealsoforminhibitorsandIgEresponsesduringfactor replacementtherapy,resultinginanaphylaxisafterrepeatedintravenousinjectionsofF.IXprotein[4,55]. However,optimalhepaticgenetransferwithAAVvectors inducestolerancetohF.IXinthisstraindespitethegene deletionmutation[4,56,57].Amongthe3othermutations thatweexamined(withendogenousnon-functionalhF.IX expressioninhepatocytes;2missenseand1nonsense mutation),theLSmutation(latestopcodon)wastheleast tolerantandwasstillpronetoantibodyresponsesto hF.IXaftermusclegenetransferusinganssAAV2vector. Interestingly,noCD8+TcellresponsewasobserveddespitelackofexpressionoftheC-terminusofhF.IXthat containstheimmunodominantCD8+Tcellepitopefor thisstrain[3].Giventhatournovelandpublisheddata demonstratedanincreasedabilityofscAAVvectorsto generatevigoroustransgeneproduct-specificCD8+Tcell responses,wehypothesizedthatamorepotentscAAV1 vectormayyieldsucharesponseintheLSstrain.Inspite ofthis,noCD8+Tcellresponseorantibodyresponsewas observedregardlessofwhetherssorscAAV1vectorwas used.Together,resultsinnullandLSmutationsshowthat theunderlyingmutationisastrongerdeterminingfactor intheriskofimmuneresponsestohF.IXthanthetypeof AAVvectorgenome.Theincreasedimmunogenicityof thescAAVvectordidnotbreaktolerancetohF.IXinthe LSmice,whichdoexpressthedominantCD4+TcellepitopeandmaythereforeexhibittoleranceintheThelper cellcompartment.Acomparisontoourpublisheddata furthersuggeststhatuseofAAV1vectorreducesantibody responsestohF.IX,atleastinmice,whencomparedto AAV2[3].Atleastequallyandperhapsmoreimportant thantheunderlyingmutationistherouteofvectoradministration/targettissue,withoptimizedhepaticgene transferresultingintoleranceinductionevenfornull mutations. Asomewhatcuriousresultoftheexperimentsinthe tolerantLSstrainwerethehigherlevelsofcirculating hF.IXachievedwiththessAAVvector.Usingtheidenticaldoseandvectorpreparations,scAAVvectoroutperformedssAAVuponmusclegenetransferinimmune deficientmice(RAG-deficientC57BL/6),whichhowever werenotavailableonastrain-matchedC3H/HeJgenetic background.Itispossiblethattheincreasedinnate immuneresponsesinducedbyscAAVvectorscouldbe silencingexpressionofthetransgene,whichmaybe strain-specific.ItisknownthattheactivityoftheCMV enhancer/promoterusedinthesevectorscanbeinhibitedbyinflammatorycytokines[58,59].IL-12-mediated inflammationatthetimeofgenetransferhasalsobeen showntoinhibittransgeneproduction[60].Similarly, theexpressionofHIVgagp24andinductionofgagspecificCD8+Tcellswaspreviouslyshowntobelower atadoseof1011than1010vg,aphenomenonwhich mayhavealsobeenrelatedtosilencingoftheCMVpromoter,orsaturationofthetransductioncapacityofthe injectedmuscleatadoseof1010vg[50].Althoughwe previouslyfoundthatIFNIinducedbyrecombinant adenovirusbutnotbyscAAVcausedtransgenesilencing, atransthyretinratherthanaCMVpromoterwasusedin thescAAVvectorsinthatstudy[61].Clearly,thereare stillfactorsaffectingtransgeneexpressionfromscAAV vectorsthatremaintobeelucidated.ConclusionInsummary,whenperforminggenetransferwithAAV vectorsviaarouteofadministrationthatismoreprone toimmuneresponsestothetransgeneproduct,theunderlyinggeneticdefectisanimportantdeterminantof theriskofBandTcellresponses.Shouldanimmuneresponseensue,whichmaybemorelikelytooccurwhen treatinginthecontextofanullmutation,scAAVvectors arelikelytocauseamorepotentCD8+Tcellresponse thanssAAV,therebyincreasingtheriskoflossoftransducedcells.Theseobservationslikelyapplytogenetherapiesforothergeneticdiseasesandshouldbetaken intoconsiderationduringclinicaltrialdesign.Abbreviations hF.IX: HumanfactorIX; F9 :FactorIX;AAV:Adeno-associatedvirus; ssAAV:Single-strandedAAV;scAAV:Self-complementaryAAV;CTL:Cytotoxic Tlymphocyte;HBmice:HemophiliaBnull-mutationmice;LS:Late-stop codonhemophilicmice;BU:Bethesdaunit;RAG:Recombination-activating gene;ELISA:Enzyme-linkedimmunosorbentassay;ELISPOT:Enzyme-linked immunosorbentspotassay;DAPI:4 ,6-diamidino-2-phenylindole. Competinginterests RWHhasbeenreceivingroyaltypaymentsfromGenzymeCorp.forlicenseof AAV-FIXtechnology. Authors ’ contributions GLR,ATM,andIZperformedexperiments.GLR,ATM,HCE,andRWH designedexperiments.GLR,ATM,HCE,andRWHinterpreteddata.HCEand RWHsupervisedandcoordinatedthestudy.GLR,HCE,andRWHwrotethe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements ThisworkwassupportedbyNationalInstitutesofHealthgrantsP01 HD078810[toRWHandHCE]andR01AI51390[toRWH].GLRwassupported byaDean  sFellowshipfromtheUniversityofFloridaCollegeofMedicine. Authordetails1DepartmentofPediatrics,DivisionofCellularandMolecularTherapy, UniversityofFlorida,Gainesville,Florida,USA.2TheWistarInstitute, Philadelphia,Pennsylvania,USA. Received:19December2013Accepted:23January2014 Published:25January2014 References1.HighKA: Thegenetherapyjourneyforhemophilia:arewethereyet? Blood 2012, 120: 4482 … 4487. 2.MingozziF,HighKA: Therapeuticinvivogenetransferforgeneticdisease usingAAV:progressandchallenges. 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MolTher 2013, 21: 796 …805.doi:10.1186/1479-5876-12-25 Citethisarticleas: Rogers etal. : Roleofthevectorgenomeand underlyingfactorIXmutationinimmuneresponsestoAAVgene therapyforhemophiliaB. JournalofTranslationalMedicine 2014 12 :25. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Rogers etal.JournalofTranslationalMedicine 2014, 12 :25Page10of10 http://www.translational-medicine.com/content/12/1/25