Human Immune Responses to Adeno-Associated Virus (AAV ...

Adeno-associated virus (AAV) is a small (25 nm), non-enveloped virus composed by an icosahedral capsid that contains a single-stranded, 4.7-Kb ... DownloadArticle DownloadPDF ReadCube EPUB XML(NLM) totalviews ViewArticleImpact SHAREON MoriyaTsuji ColumbiaUniversityIrvingMedicalCenter,UnitedStates ChengwenLi UniversityofNorthCarolinaatChapelHill,UnitedStates NathalieClement UnicornConsultations,UnitedStates Theeditorandreviewer'saffiliationsarethelatestprovidedontheirLoopresearchprofilesandmaynotreflecttheirsituationatthetimeofreview. Abstract Introduction HumanImmuneResponsestoWild-TypeAAV Post-TreatmentImmuneResponsesAgainstAAVVectors ImmuneResponsesAgainsttheTransgeneProduct ImmuneResponsestorAAVVectorsinClinicalTrialsAfterIntravenousInfusionofrAAVVectors Conclusion AuthorContributions Funding ConflictofInterest Acknowledgments References Checkforupdates Peoplealsolookedat REVIEWarticle Front.Immunol.,17April2020Sec.VaccinesandMolecularTherapeutics https://doi.org/10.3389/fimmu.2020.00670 HumanImmuneResponsestoAdeno-AssociatedVirus(AAV)Vectors GiuseppeRonzitti1*,David-AlexandreGross1andFedericoMingozzi2* 1INTEGRARE,Genethon,Inserm,UnivEvry,UniversitéParis-Saclay,Evry,France 2SparkTherapeutics,Philadelphia,PA,UnitedStates Recombinantadeno-associatedvirus(rAAV)vectorsareoneofthemostpromisinginvivogenedeliverytools.SeveralfeaturesmakerAAVvectorsanidealplatformforgenetransfer.However,thehighhomologywiththeparentalwild-typevirus,whichofteninfectshumans,poseslimitationsintermsofimmuneresponsesassociatedwiththisvectorplatform.Bothhumoralandcell-mediatedimmunitytowild-typeAAVhavebeendocumentedinhealthydonors,and,atleastinthecaseofanti-AAVantibodies,havebeenshowntohaveapotentiallyhighimpactontheoutcomeofgenetransfer.WhileseveralfactorscancontributetotheoverallimmunogenicityofrAAVvectors,vectordesignandthetotalvectordoseappeartoberesponsibleofimmune-mediatedtoxicities.Whilepreclinicalmodelshavebeenlessthanidealinpredictingtheoutcomeofgenetransferinhumans,thecurrentpreclinicalbodyofevidenceclearlydemonstratesthatrAAVvectorscantriggerbothinnateandadaptiveimmuneresponses.DatagatheredfromclinicaltrialsofferskeylearningsontheimmunogenicityofAAVvectors,highlightingchallengesaswellasthepotentialstrategiesthatcouldhelpunlockthefulltherapeuticpotentialofinvivogenetransfer. Introduction Adeno-associatedvirus(AAV)isasmall(25nm),non-envelopedviruscomposedbyanicosahedralcapsidthatcontainsasingle-stranded,4.7-KbDNAgenome.AAVsareDependovirus,astheyreplicateonlyinthepresenceofhelpervirusessuchasadenovirus,herpesvirus,humanpapillomavirusandvacciniavirus(1–4).AAVgenomeiscomposedbytwogenesrepandcap,flankedbytwopalindromicinvertedterminalrepeats(ITR).RepencodesforproteinsinvolvedinreplicationoftheviralDNA,packagingofAAVgenomes,andviralgenomeintegrationinthehostDNA(5).Capencodesforthethreeproteinsthatformthecapsid,VP1,2and3,andfortheassemblyactivatingprotein(AAP)andthenewlyidentifiedMAAP(5,6).Wild-typeAAVsnaturallyinfecthumansaround1to3yearsofage(7–9)andarenotassociatedwithanyknowndiseaseorillness(10).Afterinfection,AAVremainslatentinintegratedornot-integratedforms,untilahelpervirusprovidesthefunctionsnecessaryforitsreplication(5).InrecombinantAAVvectors(rAAV),theparentalvirusrepandcapgenesarereplacedwiththeDNAofchoiceflankedbythetwoITRs,andreferredtoasthetransgeneexpressioncassettewhenusedforgenetherapypurposes.rAAVvectorsareproducedefficientlybyseveralapproaches:transientdoubleortripletransfectionofmammaliancells(11,12);infectionofmammaliancelllineswithadenovirus(13)orherpessimplexvirus(14,15);andinfectionofinsectcellswithbaculovirus(16).Duringpackaging,repandcapgenesareprovidedintranstogetherwiththeadenoviralhelperproteinsrequiredforAAVgenomereplicationandpackaging(17,18).TripletransfectionofHEK293cellsisoneofthemostcommonlyusedmethodsforrAAVproduction.Itisbasedontheco-transfectionofthreeplasmids:onecontainingthetransgeneexpressioncassetteflankedbytheviralITRs,asecondpackagingplasmidexpressingtherepandcapgenesandathirdplasmidencodingforadenoviralhelpergenes(17,19).Historically,thepurificationofrAAVvectorswasperformedbyultracentrifugationintwosuccessivedensitygradients(17).Nowadays,thepurificationofAAVcapsidsbyaffinitychromatographyismorefrequentlyusedascolumnprocessismorescalableandyieldshighpuritypreparationsthatareamenableforclinicaluse(20).Basedonthepurificationmethod,rAAVpreparationsdifferintermsofcontaminantsandtheratioofemptytofullcapsids.AnimportantfocusinthefieldisthecontinuousimprovementoftherAAVmanufacturingprocessestoincreasevectoryieldsandpuritywhilereducingcosts(17,18,21,22).AsignificantconcernrelatedtothemethodsofproductionandpurificationistheimpactofrAAVpurityontheoverallvectorimmunogenicityprofile.OneobviousexampleofcontaminantisthepresenceofemptycapsidinrAAVpreparations(23). TheproteincapsidofrAAVaffectstheaffinityofthevectorforagiventissue.TransductionofacellbyrAAVvectorsrequirestheinteractionoftheviralcapsidwithsurfacereceptorsfollowedbyinternalizationandintracellulartraffickingthroughtheendocytic/proteasomalcompartment.Capsidproteinsthenmediatetheendosomalescapeandnuclearimport,andafteruncoating,thesinglestrandedgenomecarriedbyrAAVisconvertedtoadoublestrandedDNA.Thisconversionstepmayrepresentalimitingfactortogenetransferthatself-complementary(sc)AAVvectorscouldbypassatthecostofreducedpackagingcapacity(24).Differentfromwild-typeAAV,thegenomeofrAAVvectorsinefficientlyintegratesintothehostDNAandremainsmostlyepisomal(10,25,26).TransgeneexpressionfinallyresultsfromthetranscriptionofthemRNAandthesuccessivetranslationofthetransgenecodingsequence(Figure1)(27). FIGURE1 Figure1.Immunologicalbarrierstogenetransfer.(1)Pre-existingneutralizingantibodiestoAAVvectorsreducegenetransferefficacy.(2)CapsidscanberecognizedbyTLR2atthesurfaceofthecellsthustriggeringinnateimmuneresponses.(3)Afterendocytosis,theviralgenomecanstimulateTLR9-mediatedinnateimmunity.(4)Transgeneexpressionmaybeassociatedtothedevelopmentofanimmuneresponsethatimpactstheoutcomeofthegenetherapy.(5)CapsidproteinscanbedegradedbyproteasomeandpresentedonMHCclassI.(6)CapsidproteinscanbepresentedonMHCclassII.(7)AfterpresentationonMHCclassI,capsid-specificcytotoxicCD8+Tcellscancleartransducedcells.(8)AfterpresentationonMHCclassII,anti-capsidhumoralresponsepreventsfurthervectorre-administration. Todate,13differentAAVserotypesand108isolates(serovars)havebeenidentifiedandclassified(5,28).TherelativelylowcomplexityofAAVbiologyfacilitatesproductionofrAAVvectorscomposedbyatransgeneexpressioncassetteflankedbytheITRsfromserotype2(29)pseudotypedintoanyoftheavailableAAVcapsidvariants(5).ThisprocessallowsacomparisonofthepropertiesconferredtorAAVsbythecapsidproteinse.g.,tissuetargeting,potencyorimmunogenicity.Inparticular,thecapsidcompositioninfluencesthefirststepsoftransductioni.e.,interactionswithreceptorsontargetcellsandimpact,post-entrytraffickingandendosomalescape.Therefore,rAAVvectorsbearingdifferentcapsidshavedifferenttransductionpotential,butalsopotentiallydifferentimmunologicalproperties(5,30).Inrecentyears,severalapproacheshavebeenusedtoenablethegenerationofengineeredrAAVvectorsandasignificantexpansionoftherAAVvectortoolkit(31–34).WhiletheincreasingknowledgeonrAAVcapsidstructure-function(35)ledtothegenerationofcapsidvariantsbydirectmutagenesisofspecificaminoacidresidues,thedevelopmentofrAAVcapsidlibrariesandhigh-throughputscreeningmethodsresultedinthegenerationofavarietyofnovelcapsidsbydirectedevolution(31).Recently,twodifferentapproacheswerereportedtoovercomethelimitationsof“conventional”methodsofvectorevolution.BothmethodstakeadvantageofthelatestadvancementsinDNAsynthesisandsequencing,andareaimedatthereductionofthecomplexityoftheinitiallibrarytobescreenedeitherbyartificialintelligence(6)orbygraftingpeptidesderivedfromasubsetofproteinsinvolvedinspecificcellularfunctions(36).Regardlessofthemethodused,theisolationofnewrAAVvectorcapsidsrespondstoapreciseneedofincreasedtransductionefficacywithoptimizedbiodistributionandreducedimmunogenicity,atleastintermsofcross-reactivitywithpre-existingantibodies. Despitetheeffortsdedicatedtotheenhancementoftransductionefficacy,severalchallengesforclinicaluseofrAAVvectorsremain.Amongthem,vectorimmunogenicity,whichreflectstheinteractionsofrAAVswiththehostimmunesystem,isperhapsoftheutmostrelevancegivenitsimpactontheoutcomesoftreatmentintermsoftransgeneexpressiondurability,andtotheabilitytoeventuallyre-administerthevectorincaseoflossofefficacyovertime(Figure2).Here,wewilloutlinegeneralconceptonrAAVvectorimmunogenicity,andcomprehensivelydiscusstheclinicalexperienceinthecontextofsystemicvectoradministrationforliver-targetedgenetherapytrialsforhemophiliaandforthetreatmentofneuromusculardiseases. FIGURE2 Figure2.FactorsinfluencingAAVcapsidimmunogenicity.Theproteinsofthecapsid,thegenomeandthetransgeneproductarethemainpotentialimmunogeniccomponentsofAAVvectors.ProductionofdsRNAdrivenbythepromoteractivityofITRscanalsotriggerinnateimmunity.Additionalhost-dependentandvector-dependentfactorscanmodulatetheoverallvectorimmunogenicity.Thesefactorsaremostlypoorlyunderstood,althoughinnateimmunityactivatorslikeCpGandvectordoseappeartobeimportantdeterminantsofAAVvectorimmunogenicity. HumanImmuneResponsestoWild-TypeAAV HumoralImmunitytoAAV AlthoughAAVseroprevalencevariesgeographically,neutralizingantibodies(NAbs)thatrecognizevirtuallyallAAVserotypescanbefoundinalargeproportionofthehumanpopulation(rangingfrom30to60%).Successiveinfectionsmayexplainthehighprevalenceofanti-AAVNAbsinhumans(37–39),resultinginbroadcross-reactivityacrossAAVserotypesofdifferentorigins,includinghuman,mammalianorengineered.Regardlessofthegeographicregion,themostprevalentNAbsaredirectedagainstAAV2,followedbyAAV1(38).NAbsappearearlyinchildhoodwithapeakat3yearsofage,leavingashorttime-windowthatispotentiallyconvenientforgenetransferbetween7and11months,aftertheinfantloseshumoralprotectionbypassivetransferofmaternalantibodies(38). WhileproductionofAbsfromallfourIgGsubclasseshavebeenobserved,IgG1isthepredominantsubclassfoundinseropositiveindividualsand,ingeneral,titersofanti-AAVIgGantibodiescorrelatewiththethoseofanti-AAVneutralizingantibodies(40,41).Similarly,subjectsundergoinggenetransferwithAAVvectorsdevelopanti-AAVantibodiesfromallthefourIgGsubclasses(mainlyIgG1butalsoIgG2andIgG3)aswellasIgM,concurringtotheresultinghighneutralizingtiters(42).Interestingly,someindividualscarrynon-neutralizingIgGbindingtotheAAVcapsid(43).Whileevenlow-titerNAbsareassociatedwithefficientvectorneutralizationinvivo(44–46),thepresenceofbindingnon-NAbsappeartoenhancerAAVtransductionefficacy,atleastinsometissues(43).Pre-existinganti-AAVantibodiesinindividualsreceivingrAAVvectorsarebeinginvestigatedasapotentialsourceoftoxicityrelatedtocomplementactivation(47)althoughadirectinteractionof rAAVvectorswithcomplementproteinswasalsoreported(48,49). TCellResponsestoAAV EarlyclinicaltrialsofgenetransferwithrAAVdemonstratedthepotentialnegativeimpactofTcell-mediatedimmunityontheoutcomeofgenetransfer(45,50,51).Sincethen,researcheffortshavebeenfocusedonthestudyofpre-existinganti-AAVcellresponseandonthedevelopmentofmethodstodetectandmonitorcell-mediatedimmunityinAAV-basedgenetransfer(52–55). DifferentassaysweredevelopedtoestimatethefrequencyofTcellsspecificforAAVinhumans,includingIFN-γELISPOT(56–58)andflowcytometrybasedassays(52,56,58).Ingeneral,AAV-specificcellularresponsesarelessfrequentlyobservedthanhumoralresponses,probablyduetothelowerassaysensitivityandtothefactthatcapsid-reactivelymphocytesarefoundinlowfrequencyinperipheralblood.Thismayexplainwhydifferentindependentstudiesreportedalackofcorrelationbetweenpre-existingcellularandhumoralimmuneresponses(50,56,58).Indeed,efficientAAV-specificTcellsdetectioninperipheralbloodandspleenrequiresseveralroundsofinvitroexpansionwithpeptidelibrariesderivedfromthecapsidVP1(50,56).Alternatively,FACSstainingafteranAAVspecifictetramer-mediatedmagneticenrichmentcanbeusedtoincreasethedetectionofAAV-specificTcells(59).Recently,weshowedacorrelationbetweenanti-AAVantibodiesandcirculatingAAV2-specificmemoryCD8+TcellssecretingTNF-α(52),suggestingthatIFN-γ,whichiscurrentlybroadlyusedasamarkerofcapsid-specificTcellactivation,maynotbetheonlycytokinethatneedstobetrackedforimmunomonitoringingenetransfertrials. Asobservedwithhumoralresponses,capsidTcellresponsesarelessfrequentinyoungchildren(<5years)comparedtoolderhealthydonors(56,60),suggestingthatanti-AAVimmuneresponsesmayariseduringinfancyafterAAVinfection,andpersistthroughoutlifetimeasapoolofmemoryTcellsinsecondarylymphoidorgans.Consistently,differentiationmarkersmeasuredatsinglecelllevelbyflowcytometryindicatedthatthemajorityofAAV-specificTcellsfoundinhumanspresentamemoryphenotype(50,52,53,58).Afterexposuretothecapsidantigen,AAV-specificmemoryTcellsproduceIFN-γ,IL-2andTNF-α,andacquireacytotoxicphenotypemeasuredbytheexpressionofgranzymeBandCD107adegranulationmarkers(52,53,56,58).Inadditiontothat,twopatternsofcellularresponsestoAAVthataredependentontheserologystatusofpatientswererecentlyidentifiedbyourlaboratory(61).Exposureofhumanperipheralbloodmononuclearcells(PBMCs)obtainedfromAAV-seropositivedonorstocapsidepitopesinducedaneffectormemoryphenotypeinactivatedCD8+Tcells,withsecretionofTNF-α,andexpressionofgranzymeBandCD107a(52).Inseronegativepatients,transientactivationofNaturalKiller(NK)cells,butnotnaiveCD8+Tcells,wasobserved.TheroleoftheseactivatedNKcells,whichsecretebothIFN-γandTNF-αwithoutacytotoxicphenotype,inthecontextofgenetransferremainsunknown. Post-TreatmentImmuneResponsesAgainstAAVVectors InnateImmunityofrAAV VectorsderivedfromAAVareconstitutedbyaproteincapsid,whichishighlysimilar,ifnotidentical,tothatofwildtypeAAV,asingleordoublestrandedDNAgenomethatdoesnotexpressanyviralproteins,andtheinvertedterminalrepeats(ITR),GC-richregionsofthesinglestrandedgenomewithacomplexsecondarystructure.BoththecapsidandtheDNAcomponentsofrAAVmayconcurintheactivationofinnateimmunityalongwithotherhost-specificfactors(Figures2,3).Inadditiontothat,productionandpurificationofthevectorsleadtothepresenceofDNA-depletedAAVcapsids(emptycapsids)andbothDNAandproteincontaminants.Incomparisontootherbiologicaldrugssuchasmonoclonalantibodies,rAAVarequitecomplexandthepredictionoftheimmune-mediatedtoxicitiesaftervectoradministrationremainselusive,partiallybecauseofthelackoffullypredictiveanimalmodels(62–64). FIGURE3 Figure3.Immunomonitoringingenetransfer.Abroadrangeofassayscanbeimplementedforimmunomonitoringingenetransfertrials.Serumsamplesorotherrelevantsampleslikecerebrospinalfluid(CSF)canbeusedtomonitormarkersofinnateimmunityaswellastodetermineantibodytitersbeforeandaftervectoradministration.ThecellfractionofperipheralbloodisfrequentlyusedforbothBandTcellassaysbyELISPOT.MorecomplextechnologiescanalsobeusefulforexampletotrackTcellclonesviaTCRsequencing,ortodefinetranscriptomechangesatthesinglecelllevel.Additionally,highcontentflow-basedassaycanbeappliedforthesimultaneouscharacterizationofalargenumberofsurfaceandintracellularmarkers.WhilealotofinformationcanbegatheredbystudyingimmuneresponsetoAAVinperipheralblood,accesstotissuesamplescouldpotentiallyhelpbetterdefinethenatureofthelocalimmuneresponseinatransducedtissueaswellasitsimpactonvectorgenomepersistence.AsmanyquestionsremainonAAVimmunogenicity,thefieldofAAVgenetherapyresearchneedsfurthereffortstoresolvethecomplexityofcapsid-relatedimmuneresponses.Theharmonizationofpatientimmunomonitoringusingstandardguidelines,andqualitycontrolstocheckimmuneassayperformanceovertimeandacrossclinicaltrials,wouldgreatlyfacilitatethecomparisonofdata,andsubsequentlytheunderstandingofthecomplexityofanti-AAVimmuneresponses. Inrecentyears,significantresearcheffortswerefocusedonestablishingacausativeroleofinnateimmunityintheimmune-mediatedtoxicitiesobservedinhumans.However,theintrinsiccharacteristicsoftheinnateimmunesystem,together,andthelackofclinicalevidenceofinnateimmunesystemactivationstillrepresentachallengetowardtheappraisaloftheinnateimmunityroleintheimmune-mediatedtoxicitiesobservedingenetherapytrials. Innateimmunityisthefirstbarrieragainstpathogensasitmountsrapidlyanddoesnotrequireaspecificadaptationtothepathogens.Innateimmuneresponsedependsontherecognitionofpathogen-associatedmolecularpatternsbythepatternrecognitionreceptors(PRRs)expressedbyimmunecells.Themolecularrecognitionofviralnucleicacids,membraneglycoproteins,orevenchemicalmessengersbyPRRsleadstothenucleartranslocationofNuclearFactorκB(NF-κB)andInterferon-RegulatoryFactor(IRF),transcriptionfactorswithacentralroleintheexpressionofpro-inflammatorycytokines,ortypeIinterferons(IFNs),respectively(65). InthecontextofrAAV-mediatedgenetransfer,preclinicalstudiessupportedtheimportantroleoftypeIIFNsintheinductionofCD8+Tcellresponses.Inparticular,blockingtheactivationofinnateimmuneresponsespreventedbothcytotoxic(66,67)andhumoral(52)anti-capsidresponsesinvivo. MostofthedataontheroleoftypeIIFNsinrAAVvectorsimmunogenicitywereobtainedinthecontextofliver-targetedgenetransfer.Theliverrepresentsauniqueimmunologicalenvironment,characterizedbythepresenceofresidentimmunecellstogetherwithbothspecializedandnon-specializedantigenpresentingcells(APCs).Innon-parenchymallivercells,includingKupffercellsandliversinusoidalendothelialcells(LSECs),innateimmunityactivationafterlivergenetransferwithrAAVmainlyoccursthroughbindingtoTLR2expressedonthecellsurface(68).TherAAVdouble-strandedDNAgenome,andinparticularitsunmethylatedCpGmotifs,mayberecognizedbytheendosomalTLR9ineitherKupffercells(69),peripheralplasmacytoidDCs(pDCs)(66,70)ormonocyte-derivedDCs(71).TLR9engagementwasassociatedwithenhancedactivationofAAV-specificCD8+TcellduetoincreasedantigenpresentationonclassImajorhistocompatibilitycomplex(MHC)(54,66,72). Anintriguinghypothesisisthat,inadditiontothevectorDNAgenome,double-strandedRNA(dsRNA)mayparticipatetotheinductionofinnateimmunitytorAAV(73).Accordingtothisstudy,dsRNAsareproducedbythepromoteractivityoftheITRs.AccumulationofdsRNAswould,inturn,stimulatestheMDA5sensorinhumanhepatocytestransducedwithAAV,leadingtotheexpressionoftypeIIFNs.Interestingly,theblockadeofMDA5decreasedtheIFNresponseandimprovedtransgeneexpressionintransducedcellsinvitro(73).Althoughthishypothesisisnotyetsupportedbyclinicaldata,itmayexplainwhycellularresponsesaresometimesinitiatedweeksaftervectoradministrationinclinicaltrials,atimeframethatisconsistentwiththedsRNAsynthesisinvivo(51,74). AdaptiveImmuneSystemActivationFollowingrAAVAdministration Theinductionofanadaptiveimmuneresponserequiresalongertimethaninnateimmunityandisconsideredasthesecondbarriertowardpathogens.Ontheotherhand,adaptiveimmuneresponsesareantigen-specificandeliminatepathogenswhilegeneratinganimmunologicalmemory. TandBlymphocytesareactivatedafterthemolecularrecognitionofanantigenpresentedbyAPCs(75).Afteractivation,lymphocytesexpandanddifferentiateintoeffectorcellsandspecificallyinactivateorclearantigensthroughtheinductionofhumoralorcytotoxicresponses.Whenthelevelsofcirculatingantigenarereducedbythemountingimmuneresponse,memoryTandBlymphocytesaregeneratedandcanrespondtosuccessiveantigenicstimulationinamoreefficientandfastermanner(75).AfterrAAVvectoradministration,bothtransducedcellsandprofessionalAPCspresentcapsid-derivedepitopestocytotoxicCD8+TcellsviaMHCclassI(50,56,66,76).ActivatedCD8+TcellsmayclearrAAV-transducedcellsthusinducinginflammationinthetargetorgan,affectingthegenetransferoutcome(45,51,77,78).Theconcurrentpresentationofcapsid-derivedMHCclassIIepitopesbyprofessionalAPCsactivatesCD4+Thelpercells,whichfacilitatehumoralandcell-mediatedimmuneresponses(67).Indeed,experiencefromclinicaltrialsindicatethatrAAVvectorsadministrationleadstothedevelopmentofanti-AAVIgGandNAbs(42),likelypreventingvectorreadministration.Administrationofimmunomodulatoryregimens(79)or,B-celldepletionpriortogenetransfer,(80)havebeeneffectiveinblockinghumoralimmuneresponsestorAAVinthepreclinicalsetting.Tothisaimrituximabincombinationwithrapamyciniscurrentlybeingtestedinhumansasastrategytoenablevectorre-dosing(NCT02240407). Clinicalexperienceindicatesthat,tosomeextent,rAAVvectorimmunogenicityisdose-dependent(81,82).Lowvectordosesappeartobemanaged,inmostcases,byshortcoursesofcorticosteroidsorothermildimmunosuppressiveregimenswithrescueoftransgeneexpression(82).Accordingly,adose-dependentincreaseinAAV-antigenpresentedonMHCclassItogetherwithhigherCD8+Tcellsactivationwasreportedinvitro(76,83).Nevertheless,deleteriouseffectsofanti-capsidcellularresponse,andinparticulartheveryslowonsetoftheTcellresponse,arenotfullyrecapitulatedbyanimalmodels.ThishaspreventedtheinvestigatorstoformulatepredictivemodelsofrAAVvectorimmunogenicity,andtosomeextenthinderedthedevelopmentofspecificimmunomodulatoryprotocolsspecificforgenetransfer. ImmuneResponsesAgainsttheTransgeneProduct Alargepartofthegenetherapystrategiesformonogenicdiseasesaimatthereplacementofamutatedgenewithacorrectedcopytorestoreitsfunctionandcorrectthediseasephenotype.Thepotentialdevelopmentofanimmuneresponseagainstthetransgeneisdependentonseveralvariables,includingthetissuetargetedwithgenetransfer,thehostgeneticbackground,andtheextentoftheresidualexpressionofthedonatedgene.Genetransferinacontextofmissensemutationsandresidualendogenousexpressionofthefulllengthproteinisunlikelytoinduceanti-transgeneimmuneresponses,althoughsomepreclinicalstudiessuggestthatevensingleaminoacidvariationscanberecognizedbythehostimmunesystem(84).Conversely,genetransferinthecontextofstopmutationswithnoresidualproteinexpression,ispotentiallymorelikelytoresultinanti-transgeneimmunityduetotheabsenceofcentraltoleranceagainstthetransgeneproductitself. Intheclinicalsetting,anti-transgeneimmuneresponsesweredocumented,sofar,inonlyfewclinicaltrials,mostlyafterintramusculardeliveryofrAAVvectors.Inparticular,evidenceofTcell-mediatedanti-transgenecytotoxicTcellresponseswasdocumentedinaphaseI/IItrialofintramusculargenetransferinDuchennemusculardystrophypatients(85).Inthistrial,AAV-mediatedtransferofamini-dystrophintransgeneresultedinpoorexpressionandwasassociatedwiththedevelopmentofTcellresponsesdirectedagainsttransgeneepitopesor,possibly,intherecallofpre-existinganti-dystrophinTcellsresponse.Similarly,decreasedtransgeneexpressionandtransgene-specificcytotoxicTcellswerereportedafterintramusculardeliveryofalpha-1antitrypsinwithrAAVvectorinonesubject(86),althoughmostoftheclinicaltrialparticipantsachievedlong-termexpressionofthetransgene(87).Inotherclinicaltrials,theimpactofimmuneresponseonthetreatmentoutcomeswaslessclear.Finally,inaphaseI/IItrialofintracranialdeliveryofarAAV5vectorformucopolysaccharidosistypeIIIB,anti-transgeneTcellswerealsoreported(88).Takentogether,theclinicaldatapresentedsuggestthatdisease-specificconditionse.g.,theongoinginflammationinmuscledystrophies(85),arelikelytoincreasetransgeneimmunogenicityaftergenetransfer. Theapparentinconsistencybetweenthepotentialimmunogenicityofthetransgeneandthelownumberofactualreportsofanti-transgeneimmunogenicresponsescouldpossiblybeexplainedalsobythefactthatmostoftheclinicaltrials,sofar,wereperformed:(i)insubjectsalreadyexposedtoproteinreplacementtherapypriortogenetransfer;(ii)insubjectswithresidualendogenousexpressionofthegenetargetedwithgenetransfer;(iii)bygenetransferrestrictedtoimmune-privilegedcompartmentsliketheeye,theliverorthebrain;and(iv)whengenetransferwasadministeredtogetherwithanimmunomodulatoryregimen. Oneimportantdeterminantofanti-transgeneimmuneresponsesisthetissuedistributionoftransgeneexpression.TheselectivityofgeneexpressionforagiventargettissueistheresultofthecombinedtropismoftheAAVcapsid,therouteofvectoradministrationandthespecificityofthepromoterincludedinthetransgeneexpressioncassette.Ingeneral,intramuscularadministrationandstrongubiquitouspromotersaremorelikelytoinduceanti-transgeneimmuneresponsesthansystemicadministrationandtissue-specificpromoters(89,90).Anotherlayerofcomplexityintheevaluationofthepotentialimmunogenicityofgenetransferisthat,insometissues,suchasmuscle(91),thepresenceofinflammationduetotheunderlyingdiseasemightresultinahigherriskoftriggeringtransgene-directedimmuneresponses.Conversely,intissuesthataredefinedasimmune-privilegedperseduetothepresenceofbarriersthatreduceantigenpresentationandimmunesystemcellstrafficking(e.g.,theeyeorthenervoussystem)ortotheirparticularimmunologicalmilieu(e.g.,theliver),theoverallriskofencounteringanti-transgeneimmuneresponsesislow. Theliver,duetotheconstantexposuretonon-selfantigens,haspeculiarimmunologicalpropertiesthatpreventuncontrolledimmuneactivation.SeveralstudiesofgenetransferwithrAAVinbothsmallandlargeanimalsindicatedthathepatocyte-restrictedtransgeneexpressioninducedarobust,antigen-specificperipheraltolerance(60,92,93).Inanimalmodels,liver-inducedimmunologicaltolerancehasbeenexploitedtocounteractdeleteriousimmuneresponsesinducedbygenetransfertargetingmoreimmunogenictissues,suchasthemuscle(89,94).Thedifferentantigenpresentingcells(APCs)residentintheliverareinvolvedinthetolerogeniceffectafterlivergenetransfer.Inparticular,Kupffercells,themacrophagesresidentintheliver,seemtohavealessmaturephenotypecomparedtootherprofessionalAPCs(95,96).This,togetherwiththesecretionoftheanti-inflammatorycytokineIL-10(97–99)byKupffercellsleadstopoorTcell-activation.AntigenpresentationthroughMHCclassIexpressedontohepatocyteshasbeenassociatedwithincompleteCD8+Tcellactivationandincreasedexhaustionandapoptosis(89,93,94,100–103).Liversinusoidalendothelialcells(LSECs)canalsoactasprofessionalAPCsandpromotetolerancethroughtheinductionofTregulatorycells(Tregs)(104,105).TregsplayanessentialroleintoleranceinductionafterlivergenetransferasdemonstratedbytheincreasedtransgeneimmunogenicityobservedafterTregsdepletion(60,89,92,106).Consistently,increasedTregsexpansionbyrapamycintreatment,favoredtheinductionofliver-mediatedtoleranceeveninthepresenceofpre-existinganti-transgeneimmunity(107,108).OthermechanismsliketheinductionofCD8+regulatoryTcells(109),thedegradationofTcellsinhepatocytes(110),andtheCD4+Tcellanergy(111)wereproposedintheestablishmentandmaintenanceoflivertolerance. ImmuneResponsestorAAVVectorsinClinicalTrialsAfterIntravenousInfusionofrAAVVectors LiverGeneTransfer–TheExperienceWithHemophiliaB ThelargestsetofclinicaldataavailableonrAAV-mediatedgenetransferforthetreatmentofliverdiseasesderivesfromhemophiliaBstudies.HemophiliaBisanidealtargetforrAAVgenetherapyfordifferentreasons.First,thetransgene,humancoagulationfactorIX(hFIX),canbeexpressedinavarietyoftissuesincludingmuscleandliver,thelatterbeingitsnaturalsiteofsynthesis,andlowlevelsoftransgeneexpression(around5%ofnormal)aresufficienttogreatlyreducetheimpactofthediseaseonthequalityoflifeofthepatients.AnotherimportantadvantageisthathFIXissmalland,basedontheexperiencewithproteinreplacementtherapy,seemstohavearelativelylowerimmunogenicitypotentialcomparedto,forexample,humancoagulationfactorVIII.Finally,thediseaseisverywellcharacterized,smallandlargeanimalmodelsofhemophiliaBareavailable,andmethodsandendpointstoevaluatetheefficacyofagiventreatmentarewellestablished. ThefirstdemonstrationthathFIXcanbesecretedbyhumanhepatocytesfollowingrAAVvector-mediatedgenetransferwasobtainedinaseminalclinicaltrialwhere7subjectswithseverehemophiliaBreceivedthroughthehepaticarteryasingle-strandedrAAV2vectorcarryingthehFIXtransgeneunderthecontrolofaliver-specificpromoter(45).Theclinicaltrialwasdesignedwiththreeincreasingdosecohortsof8×1010vg/kg,4×1011vg/kg,and2×1012vg/kg,respectively.TherapeuticlevelsofhFIXexpressionwerereportedonlyinthefirstpatientwhoreceivedthehighestvectordose.However,differentlyfromanimalmodelsthatshowedastableexpressionofhFIXovertime(51,112),inhumans,transgeneexpressionstartedtodecline4weeksaftervectorinjection.Thisdeclinewasassociatedwithaself-limitedincreaseinlivertransaminasesandthedetectionofcirculatingAAV-specificCD8+Tcells(45,50).Inasecondpatientdosedatthe2×1012vg/kgdose,notransgeneexpressionwasobservedpossiblyduetothepresenceofananti-AAV2pre-existinghumoralimmuneresponse(45). ThisfirstclinicaltrialdemonstratedthattherAAVvectorsweresafeandefficaciousinlivertargeting,althoughtransgeneexpressionwasonlytransient.Bothsmallandlargeanimalmodelsusedinpreclinicalresearchfailedtopredictthisnegativeoutcomelinkedtoananti-AAVcapsidcellularresponse.Nevertheless,thisclinicaltrialprovideduniqueinformationforthefutureuseoftherAAVtechnologyforgenetransferinhumans.AsecondclinicaltrialwasthencarriedoutusingrAAV8serotypefortheexpressionofhFIX(81).Theimprovedtransductionofhepatocytesachievedwiththisserotype(113)wascombinedwithaself-complementarygenomeandacodon-optimizedtransgenesequencetooptimizetheexpressionintheliver(114). Inthissecondtrial,participantswerescreenedforpre-existinghumoralresponseagainstAAV8andonlyseronegativepatientswereincluded.Threedosesofthevectorwereinfusedthroughaperipheralveinrangingfrom2×1011vg/kgto2×1012vg/kg.Differentlyfromtheprevioustrial,hFIXexpressionwasdetectableandreached1–4%ofnormalinthelowandmiddosecohorts(81).Atthehighestdose,inthefirstsubjectdosedfromthiscohort,8weekspost-infusionthetherapeutichFIXlevels(approximately8–10%ofnormal)startedtodeclineand,similarlytotheprevioustrial,anelevationofliverenzymesandanincreaseincirculatingcapsid-specificTcellswasdetected.Ataperingcourseofsteroidswasadministeredtocontroltheliverenzymeelevation,whichallowedforrescueoftransgeneexpression(81).Oftheadditionalparticipantsenrolledinhighdosecohort,atotalof6,4developedatransienttransaminitisthatrapidlyresolvedaftertransienttreatmentwithprednisolone(74).Inthisstudy,inmostparticipants,andinparticularthosefromthehighdosecohort,asignificantreductioninannualizedbleedingepisodesintheabsenceofrecombinanthFIXprophylaxiswasreported(74)withastabletransgeneexpressiondocumentedforupto10years(115). ResultsobtainedinathirdclinicaltrialforhemophiliaBfurthersupporttheroleofthetotalcapsiddoseasadeterminantofrAAVvectorimmunogenicity.Subjectsseronegativeforanti-AAVantibodiesreceivedarelativelylowdose(5×1011vg/kg)ofanAAVvectorexpressingahyperactivevariantofhFIX(hFIX-R338L).Atthisdose,only2outof10participantshadanelevationofliverenzymes,whichwassuccessfullycontrolledwithcorticosteroids.InthisstudyrAAVadministrationresultedintherapeutictransgeneexpressioninallenrolledsubjects(78). rAAV5wasalsousedtoexpresshFIXinhepatocytesofhemophiliaBpatients.Thisvector,producedinabaculovirussystem,wasinjectedatdosesupto2×1013vg/kg,in10hemophiliaBpatients(116).TherapeuticlevelsofhFIXwereobservedformostofthetreatedpatients.ALTelevation,whichwasreportedin3outof10patients,wasneitherassociatedwithTcellsactivationdetectedbyELISPOT,norcorrelatedwiththepresenceofpreexistingNAbs,andwastreatedbycorticosteroidswithnomeasurabledecreaseinhFIXtransgeneexpression.Similarly,inaclinicaltrialforhemophiliaA,theinfusionofupto6×1013vg/kgofarAAV5vectorexpressinghumancoagulationfactorVIIIresultedinALTelevationinseveralenrolledsubjects(117).Theimpactofliverenzymeelevationontransgeneexpressioninthisstudyisbeingdebated,althoughadecreaseinfactorVIIIlevelshasbeendetectedinseveralparticipantsfollowedatlong-term(118). CorticosteroidtreatmentgiveninresponsetoALTelevationusuallycontrolledtheanti-AAVvectorimmuneresponseandstabilizedtransgeneexpression.However,insomecasesthisapproachhasfailedtocontroltheanti-capsidimmuneresponses.Oneexampleisaclinicaltrialinwhich7hemophiliaBsubjectsreceivedascAAV8vectorexpressingthehyperactivehFIX-R338Lvariant(119)atdosesrangingfrom2×1011vg/kgto3×1012vg/kg(NCT01687608).Sustainedtransgeneexpression(hFIXactivityofabout20%)wasreachedinonlyoneparticipant,whileallotherpatientsenrolledinthetrialhadeithernoexpressionorlosttransgeneexpressiondespitecorticosteroidtreatmentwithin5to11weekspostvectorinfusionwithoutanyevidenceofanti-hFIXantibodiesformation(120).VectorimmunogenicitywaspossiblydependentontheelevatedCpGcontentofthetransgeneexpressioncassette.Indeed,transductionofprimaryhumanlivercellswiththisvectorinducedthesecretionofmoreTh1-orientedchemokinescomparedtoaCpG-nullversionofthesamevector(121).Inasecondclinicaltrial,rAAVRh10serotypeexpressinghFIXwasadministeredtosixseronegativehemophiliaBpatientsatdosesof1.6×1012and5×1012vg/kg(122).AtransientexpressionofhFIXwasreportedalsointhisstudy.Aftervectoradministration,5outof6injectedindividualshadALTelevationassociatedwithlossofthetransgeneexpressiondespitecorticosteroidtreatment.Fourofthemdemonstratedlowanti-capsidandanti-hFIXresponseasmeasuredbyIFN-γELISPOTassay,possiblyreflectingthehighdosesofcorticosteroidstheyreceived.Subject6hadahigherALTelevation,associatedwithastrongCD4+IL-2+IFN-γ+TcellresponseagainstanepitopespanningthehFIXmutation,andincreasedinflammatorycytokinesintheserum(123),however,noanti-hFIXhumoralimmuneresponsewasdocumentedinthissubject. Takentogether,theclinicalexperienceaccumulatedwithclinicaltrialsforHemophiliaBindicatethatmildimmuneresponsestothevectormaycleartransgeneexpressionfromtheliver.Theseimmuneresponsesarecontrolledbycorticosteroidtreatmentinmostofthecases.However,transientexpressionofthehFIXtransgenewasobservedregardlessofcorticosteroidtreatmentinsomeclinicaltrials,potentiallyduetoahigherintrinsicimmunogenicityofthevectorsused. AAVGeneTransferforNeuromuscularDiseases Neuromusculardiseasesofgeneticoriginareamultitudeofdiseasesthatareheterogeneousintermsofpathophysiology,tissuesinvolved,ageofonset,andclinicalmanifestations.ClinicalexperienceaccumulatedongenereplacementwithrAAVvectorsindicatesthatregardlessofthedisease,muscleorcentralnervoussystemtargetingrequireshighvectordosesintherangeof1×1014vg/kg.Inrecentyears,theimprovementsinlarge-scalerAAVvectormanufacturingallowedfordeliveryofthelargedosesofAAVvectorsneededinpatientswithneuromusculardiseases. OneofthemostexcitingresultsinthefieldofrAAV-mediatedgenetransferwastherecentapprovalofZolgensma(Avexis,Novartis)forthetreatmentofspinalmuscularatrophy(SMA)typeI(124).Theefficacyofthisdrugwasinitiallyprovedinapivotalclinicaltrialinvolving15patientswithSMAtypeI(125).ArAAV9expressingtheSMN1geneunderthecontrolofaubiquitouspromoterwasinfusedintravenouslyatdosesof6.7×1013and2.0×1014vg/kg.Inthefirstpatientdosedatthelowvectordose,arobustALTelevation(31timesabovetheupperlimitofanormalrange)wasreportedalthoughitwascontrolledbycorticosteroids.Afterthisfirstobservation,prophylacticcorticosteroidstreatmentwasapplied(30days,starting1daybeforevectorinfusion)andALTelevationwasreportedonlyin3patientswithinthehighdosegroup(125).Atthesametime,highpercentageofcapsid-specificTcellsweredetectedinperipheralbloodbyIFN-γELISPOT(126).However,neithertheALTelevationnorthepresenceofcapsid-specificTcellswasassociatedwithreducedvectorefficacy.Theexcellentefficacyprofileandthemildadverseeventsreportedinthisfirstclinicaltrialswereconfirmedatlong-term(125,127,128)andalsoinaphaseIIItrial(ClinicalTrials.gov:NCT03306277).Basedontheseresults,adose-findingclinicaltrialwaslaunchedtoevaluatethesafetyandefficacyofintrathecaladministrationofZolgensmainSMAtype2patients(ClinicalTrials.gov:NCT03381729).Sofar,31patientsreceivedthetreatmentatthethreevectordosesrangingfrom6×1013to2.4×1014totalvectorgenomes(129).Resultsofthefirsttwodosecohortsindicatedanameliorationofthemotorfunctionwithmildadverseevents.Despitecorticosteroidstreatment,elevatedALTandASTwerereportedinonepatientandwereconsideredrelatedtothetreatment.However,thistrialwasplacedonpartialholdbytheFDAafterthedetectionofpossibletoxicityindorsalrootganglianeuronsintwoindependentpreclinicalstudiesperformedinlargeanimals(130,131).Importantly,thisunexpectedtoxicitywasneverreportedinpatientsthatreceivedthesamevectorbyperipheralvein(125)orinadifferenttrialofrAAVgenetransferforgiantaxonalneuropathy(ClinicalTrials.gov:NCT02362438)inwhichintrathecaldeliveryofrAAV9wasassociatedwithtransientcorticosteroidsandalongercourseoftacrolimusandrapamycin. AdditionalinformationontheadministrationofhighdosesofrAAVinpediatricpatientsderivesfromaPhaseI/IIclinicaltrialforX-linkedmyotubularmyopathy(MTM,ClinicalTrials.gov:NCT03199469)andthreestudiesforDuchennemusculardystrophy(DMD,NCT03375164,NCT03362502,NCT03368742).IntheMTMstudy,dosesupto3×1014vg/kgofarAAV8vectorexpressingtheMtm1transgeneunderthecontrolofamuscle-specificpromoterwereadministeredtopatientslessthan5yearsold(132).Clinicallymeaningfulimprovementtogetherwithrobustproteinexpressionandrecoveredhistologywerereported.Inthisstudy,aprophylacticregimenofcorticosteroidwasapplied.Althoughthevectorwasgenerallywelltolerated,increasedliverenzymes,creatininekinaseandtroponinwerereported.Bothanti-transgeneandanti-capsidimmuneresponsesweredetectedaftervectoradministration,althoughitisunclearwhetherthereisanycorrelationwiththeclinicaloutcomes.Furtherstudiesandlong-termfollow-upofparticipantswillhelptoelucidatetherelevanceofthemeasuredimmuneresponses. VariableoutcomesacrosstrialswerereportedinthecontextofsystemicrAAVvectordeliveryforDMD.OneclinicaltrialusedarAAVrh74vectortoexpressamicro-dystrophintransgenewiththeMHCK7musclespecificpromoter(ClinicalTrials.gov:NCT03375164).Inthistrial,3outof4DMDpatientswhoreceivedadoseof2×1014vg/kghadelevatedliverenzymes,whichwerecontrolledbyincreasingcorticosteroiddose(133).Sofar,nodatawasprovidedbythesponsoronspecificresponsesagainstthecapsidorthetransgene,althoughsustainedtransgeneexpression,togetherwithameliorationofcirculatingcreatinekinaselevels(133),suggestnoimpactofimmuneresponsesonmuscletransduction.Arandomized,doubleblind,andplacebo-controlledstudywasrecentlyopenedtostrengthenthedataofthispilotstudy(ClinicalTrials.gov:NCT03769116).Twoweeksaftervectorinjection,acaseofrhabdomyolysiswasreportedinaparticipantfromthissecondstudy.However,thestudydrugsafetymonitoringboardreviewedthedataandrecommendedthestudytocontinue,thussuggestingthattheadverseeventwaspossiblyunrelatedtotheinvestigationalproduct. TwoadditionalstudiesofsystemicrAAVvectordeliveryforDMDpresentedamorecomplexclinicalpicture.Inthefirstone,arAAV9vectorexpressingamicro-dystrophinunderthecontrolofamuscle-specificpromoterwasinfusedintravenouslyin6adolescentpatientsattwodoses,5×1013and2×1014vg/kg(ClinicalTrials.gov:NCT03368742).Despitemicrodystrophinexpression,thistrialwasplacedonholdbecause2patients,oneinthelowdoseandthesecondinthehighdosecohortdevelopedacutekidneyinjuryandactivationofthecomplementsystemwithsignsofcardiopulmonarydecline(134).InasecondtrialforDMD,1×1014vg/kgor3×1014vg/kgofanrAAV9vectorexpressingamini-dystrophinunderthecontrolofmuscle-specificpromoterwasinjectedin6adolescentpatientsfrom6to12yearsofage(ClinicalTrials.gov:NCT03362502).Mini-dystrophinexpressionlevelsabove20%ofwild-typewereobservedat2monthspostvectorinfusion,whileactivationoftheimmunesystem,asmeasuredbyneutralizingantibodylevelsandT-cellresponseswithELISPOT,wasdocumentedinalltheparticipants(135).Asinthepreviousclinicaltrial,oneoftheparticipantsshowedsymptomsofcomplementactivationandacutekidneyinjurythatrequiredatreatmentwithacomplementinhibitor(Eculizumab).Importantly,thesponsorobservedthattheactivationofthecomplementwasassociatedwitharapidantibodyresponseagainstthevector(135). ThedataobtainedfromclinicaltrialsinneuromusculardiseasessupporttheconceptthathighdosesofrAAVvectorsareingeneralwelltoleratedandhavethepotentialtotreatraregeneticdiseasesaffectingmuscleorcentralnervoussystem,particularlywhenthesehighdosesareadministeredduringearlychildhood.However,inadolescentpatients,complementactivation,possiblyduetoexaggeratedanti-vectorimmuneresponsesmayrepresentalimitationtotheapplicationofgenetherapyatsuchdoses.ItshouldbenotedthatthishypersensitivitytorAAVwasidentifiedinDMDpatientsthat,duetotheongoingmuscledegenerationandtheunderlyinginflammation,haveapeculiarimmunologicalenvironmentthattendstoexacerbateimmuneresponses(91).Thedisease-specificityoftheseresponsesispossiblysupportedbythefactthatinaclinicaltrialforLimb-girdlemusculardystrophytypeE,noinstancesofimmunemediatedtoxicitieswerereporteddespitetheuseofasimilarvectoratcomparabledoses.Othertriggeringfactorsarealsobeingconsideredtoexplaintheseemergingclinicalfindings,suchastheageofthesubjectsenrolled,thedifferentvectorsusedinthestudies,andeventuallycontaminantsderivedfromthedifferentprocessesusedtomanufacturetheclinicallotsofrAAVsusedinthestudies.Largerstudieswillhelptobetterdefinethedeterminantsoftheimmunotoxicitiesobservedinthosetrials. Conclusion Inrecentyears,wehaveaccumulatedsignificantclinicalexperiencewithAAVvectors.WhilethestudyofimmuneresponsesinAAVtrialshasresultedinimportantadvancesforthefield,alotmoreneedstobedonetoprovideaclearpictureofthecomplexinteractionsofAAVwiththehostimmunesysteminthedifferentclinicalsettingsofgenetransfer.Ofnote,relativelylittleisunderstoodonthehost-andvector-dependentfactorsinfluencingthedevelopmentofcytotoxicimmuneresponsesleadingtopoorgenetherapyoutcomesintermsofdurationoftransgeneexpression.Tothisend,onelimitationofthecurrentimmunomonitoringmethodsisthattheyrelyontheinvitrotestingofimmunecellsisolatedfromperipheralbloodvs.lymphocytesinfiltratingtheperipheraltissuestransducedwithrAAVvectors.Adistinctactivationprofileoftissue-residentcells,andthevariabilityassociatedtoPBMCscollectionandtestingacrossclinicaltrials(i.e.,thelackofstandardizationofassaysusedforimmunomonitoring),mayexplainthepoorcorrelationbetweenIFN-γELISPOTandimmunogenicityoutcomesobservedinsomeclinicaltrials.Additionally,themonitoringofcytokinesotherthanIFN-γmaybehelpfulinmoreeffectivelymonitorTcellactivationfollowingrAAVvectoradministration(52)(Figure3).Therelativelylownumberofsubjectsenrolledineachtrialalsorepresentsapotentiallimitationtothestudyofimmuneresponsesaftergenetransfer,althoughtheanalysisofthecollectiveresultsintheclinic,alongwithpreclinicalstudies,hasbeguntohighlightsomeofthedeterminantsofvectorimmunogenicity(e.g.,theabilityofrAAVvectorstoactivateinnateimmunity).AmoresystematicandstandardizedapproachtoimmunomonitoringinrAAVtrialsmayhelpfurtherboostourknowledgeonvectorimmunogenicityinhumans,particularlyforwhatconcernstheunderstandingoftheimportanceofthedisease-specificimmunecontextofgenetransfer.Thiswillbekeytodevisestrategiesaimedatreliablyachievingsafeandlong-lastingtherapeuticefficacyfollowingrAAVvectordelivery. 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Editedby: MoriyaTsuji,ColumbiaUniversityIrvingMedicalCenter,UnitedStates Reviewedby: NathalieClement,UniversityofFlorida,UnitedStates ChengwenLi,TheUniversityofNorthCarolinaatChapelHill,UnitedStates Copyright©2020Ronzitti,GrossandMingozzi.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(CCBY).Theuse,distributionorreproductioninotherforumsispermitted,providedtheoriginalauthor(s)andthecopyrightowner(s)arecreditedandthattheoriginalpublicationinthisjournaliscited,inaccordancewithacceptedacademicpractice.Nouse,distributionorreproductionispermittedwhichdoesnotcomplywiththeseterms. *Correspondence:GiuseppeRonzitti,[email protected];FedericoMingozzi,[email protected] Peoplealsolookedat Download