Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model

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Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model
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Grimstein, Christian
Choi, Young-Kook
Wasserfall, Clive H.
Satoh, Minoru
Atkinson, Mark A.
Brantly, Mark L.
Campbell-Thompson, Martha
Song, Sihong
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BioMed Central
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Background: Alpha-1 antitrypsin (AAT) is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT) gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD) mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). Methods: DBA/1 mice were immunized with bovine type II collagen (bCII) to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAVhAAT). Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-a family (BAFF), antibodies against both bovine (bCII) and mouse collagen II (mCII) were tested by ELISA. Results: Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8)-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially. Conclusion: These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.
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This work was supported by grants from NIH (DK58327) and University of Florida Office of Research. Publication of this article was funded in part by the University of Florida Open-Access publishing Fund.
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Journal of Translational Medicine 2011, 9:21

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RESEARCH OpenAccessAlpha-1antitrypsinproteinandgenetherapies decreaseautoimmunityanddelayarthritis developmentinmousemodelChristianGrimstein1,Young-KookChoi1,CliveHWasserfall2,MinoruSatoh2,3,MarkAAtkinson2,MarkLBrantly3, MarthaCampbell-Thompson2,SihongSong1*AbstractBackground: Alpha-1antitrypsin(AAT)isamulti-functionalproteinthathasanti-inflammatoryandtissue protectiveproperties.WepreviouslyreportedthathumanAAT(hAAT)genetherapypreventedautoimmune diabetesinnon-obesediabetic(NOD)miceandsuppressedarthritisdevelopmentincombinationwithdoxycycline inmice.InthepresentstudyweinvestigatedthefeasibilityofhAATmonotherapyforthetreatmentofchronic arthritisincollagen-inducedarthritis(CIA),amousemodelofrheumatoidarthritis(RA). Methods: DBA/1micewereimmunizedwithbovinetypeIIcollagen(bCII)toinducearthritis.Thesemicewere pretreatedeitherwithhAATproteinorwithrecombinantadeno-associatedvirusvectorexpressinghAAT(rAAVhAAT).Controlgroupsreceivedsalineinjections.Arthritisdevelopmentwasevaluatedbyprevalenceofarthritisand arthriticindex.SerumlevelsofB-cellactivatingfactoroftheTNFa family(BAFF),antibodiesagainstbothbovine (bCII)andmousecollagenII(mCII)weretestedbyELISA. Results: HumanAATproteintherapyaswellasrecombinantadeno-associatedvirus(rAAV8)-mediatedhAATgene therapysignificantlydelayedonsetandameliorateddiseasedevelopmentofarthritisinCIAmousemodel. Importantly,hAATtherapiessignificantlyreducedserumlevelsofBAFFandautoantibodiesagainstbCIIandmCII, suggestingthattheeffectsaremediatedviaB-cells,atleastpartially. Conclusion: Theseresultspresentanewdrugforarthritistherapy.HumanAATproteinandgenetherapiesare abletoameliorateanddelayarthritisdevelopmentandreduceautoimmunity,indicatingpromisingpotentialof thesetherapiesasanewtreatmentstrategyforRA.BackgroundRheumatoidarthritis(RA )isasystemicautoimmune disease,characterizedbychronicjointinflammationand synovialhyperplasialeadingtoboneandjointdestruction.Thelifeexpectancyisloweredandqualityoflifeis decreasedinRApatients.Sofarlittleisknownabout theactualdiseaseinitiatingstimulus;however,extensive researchoverthelastdecadeshaveshownthatmultiple geneticaswellasenvironmentalfactorsinteractand triggertheonsetofRA[1,2].TheautoimmuneinflammationofRAismaintainedbyinappropriateactionof macrophages,B-cells,T-cells,andothertypesofcells leadingtodysregulatedcytokine/chemokineproduction. Thesynovialinflammationi scausedbyinfiltrationand proliferationofactivatedimmunecellsincludingneutrophils,macrophages,fibroblasts,mastcells,NKcells, NKTcells,T-cellsaswellasplasmacells[3].Progressivejointandbonedestructionismediatedthroughthe activitiesofosteoclasts,ch ondrocytes,synovialfibroblastsandcytokineinductionofdestructiveenzymes, chieflymatrixmetalloprot einases(MMP)[4].Current therapymainlyaimstoinhibitthebiologicalfunctionof tumornecrosisfactor-alpha(TNFa )andlymphocyte proliferation.Duetoineffectivenessofanti-TNFa therapyincertainpatientsandvarioussideeffectsofmethotrexatewhichinhibitslymphocytesproliferation,thereis *Correspondence:shsong@ufl.edu Contributedequally1DepartmentofPharmaceutics,UniversityofFlorida,Gainesville,FL32610, USA FulllistofauthorinformationisavailableattheendofthearticleGrimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 2011Grimsteinetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.

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stilltheneedtoidentifynewtargetmolecules/pathways andtodevelopnewtreatment[5].Immunoregulatory andanti-inflammatorystrategiesthataffectB-cellactivation,T-cellactivationorinhibitproinflammatorycytokineshaverecentlyshowngreatpotentialforthe treatmentofRA[5,6]. Humanalpha-1antitrypsin(hAAT)isa52kDaserum glycoprotein,synthesizedprimarilyintheliver.Itisalso expressedinothertypesofcellsincludingneutrophils, monocytes,macrophages,alveolarmacrophages,intestinalepithelialcells,car cinomacellsandthecornea [7-10].ThenormalserumlevelofhAATis1-2mg/ml. Duringinflammation,hAATlevel,asanacutephase reactant,canincrease3-4folds,suggestinganimportant roleinrespondingtoinflammationinthehumanbody. IncreasingevidenceindicatesthathAATisimmunoregulatory,anti-inflammatoryandmaybeusedforthe treatmentofRA.Itinhibitsneutrophilelastaseandproteinase3withhighefficiency,aswellascathepsinG, thrombin,trypsinandchymotrypsinwithlowerefficiency[11].Mostoftheseproteasestargetreceptorproteins,involvedinproinflammatorycytokineexpression andcellsignaling[12].Italsohasbeenreportedthat neutrophilelastaseinhibitorsreduceincidenceaswellas severityofcollagen-inducedarthritis(CIA)inbothrats andmice[13].HumanAATisabletocompletelyeliminatetheacuteinflammatoryinfiltrationandconnective tissuebreakdowninthelunginacigarettesmokeinducedemphysemamousemodel[14].Italsoinhibits lipopolysaccharide(LPS)-stimulatedreleaseofTNFa andinterleukin(IL)-1 b ,andenhancestheproduction ofanti-inflammatorycytokineIL-10[15-17].Human AATsignificantlyprotectsa gainstthelethalityinduced byTNFa orendotoxininmice[18].Itcanalsoinduce expressionofIL1-Rainhumanperipheralbloodmononuclearcells(PBMC s)[19]andreducesischemiainducedapoptosisandinflammation[20].Wehave recentlyshown,thatcombinationtherapyusingdoxycyclineandhAATgenetherapyreducesarthritisdevelopmentinmice,suggestingatherapeuticeffectofhAAT inanarthritismousemodel[21]. Recombinantadeno-associa tedvirusvectors(rAAV) havebeenwidelyusedforgenetherapyinanimalmodelsandhumanclinicaltrials[22],becauseoftheir uniquefeaturesinsafetyandefficiency.Ithasbeen reportedthatrAAVmediatedlong-termandhighlevels oftransgeneexpressioninawidevarietyoftissues, includingmuscle[23],lung[24],liver[25],brain[26] andeye[27].RecentlydevelopedrAAVvectorsincludingnewserotypesofAAV,mutantsAAVanddouble strandedAAVhaveprovidedmoreopportunitiesand challengesfortheirapplication[28-31].Previously,we haveshownhAATgenetherapyusingrAAV2and rAAV1vectorspreventedtype1diabetes.However,the immuneresponsetothetransgeneproduct(hAAT) complicatedthetherapeuticeffect[32,33].Wehave recentlydiscoveredthatrAAV8vectorfailtotransduce dendriticcellsandinduceimmunetolerancetotransgeneproductentailingrAAV8asapromisingvector usedfortherapeuticintervention[34]. InthepresentstudywefurtherinvestigatedthefeasibilityofhAATwithitsanti-inflammatoryandimmunoregulatorypropertiesforthetreatmentofRAusingboth, proteintherapyandrAAV8mediatedgenetherapy.MethodsrAAVVectorProductionTherAAV-CB-hAATvectorconstructwasproduced andpackagedaspreviouslydescribed[27].Briefly,this vectorcarrieshAATcDNAdrivenbythecytomegalovirus(CMV)enhancerandchicken b -actinpromoter andcontainsAAV2invertedterminalrepeats(ITRs).It waspackagedintoAAVserotype8capsidbycotransfectionofvectorplasmidandhelperplasmid(XYZ8)into 293cells.rAAV8-CB-hAATvectorswerepurifiedby iodixanolgradientcentrif ugationfollowedbyanionexchangechromatography.Thephysicalparticletitersof vectorpreparationswereassessedbydotblotanalysis.AnimalsSixweek-oldmaleDBA/1micewerepurchasedfrom HarlanSpragueDawley,Inc. (Indianapolis,IN),housed inaspecificpathogen-freeroomasapprovedbythe UniversityofFloridaInstitutionalAnimalCareandUse Committee.Forinductionofa rthritis,bCII(Chondrex LLC,Redmond,WA)wasdissolvedin0.05Naceticacid ataconcentrationof2mg/mlbystirringovernightat 4CandwasemulsifiedwithanequalvolumeofCompleteFreund sAdjuvant(CFA)(ChondrexLLC,Redmond,WA).Attheageofeightweeks,DBA/1mice wereimmunizedintradermallyatthebaseofthetail with0.1mlofemulsioncontaining100 goftypeIIcollagen.Threeweeksafterpriming(day21),themice wereboostedwith0.1mlofbCII(100 g)emulsifiedin equalvolumeofincompleteFreund sAdjuvant(IFA) (Difco,Detroit,MI).Forassessmentofarthritis,allmice weremonitoredthreetimesaweekbythesameperson blindedtothetreatmentgroupandevaluatedtheincidenceofarthritisandclinicalscore.Anarthritisscore systemrangingfromstage0-4wasused:0:noswelling orredness;1:detectablearthritiswitherythema;2:significantswellingandredness;3:severeswellingandrednessfromjointtodigit;4:jointstiffnessordeformity withankylosis[35].Theclinicalscorewasexpressedas theaveragecumulativevalueofallfourpawswitha maximumscoreperanimalof16.Severearthritiswas definedasarthritisscore>3forthepurposeofcomparingdatabetweengroups.Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page2of13

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HistologicalAssessmentFortheanalysisofarthritis,micewereanesthetizedand sacrificedbycervicaldislocationonday28afterimmunization.Thetwohindlimbsofmiceintreatmentand controlgroupswereremoved.Specimenswerefixedin formalinanddecalcifiedinRDOsolution(Apex,Aurora, IL)for10-20mindependingontissuesizeandthen checkedmanuallyforpliability.Sections4 mthick werecutandstainedwithhematoxylinandeosin accordingtostandardmethods. Histologicalevaluationwasperformedbytwoindependentandblindedpathologists.Infiltrationofimmune cells,hyperplasia,pannusformationandbonedeformationwasdeterminedforeachpawusinganevaluation scalerangingfrom0-3accordingtoseverityofpathohistologicalchanges.(0:normal,1:mild,2:moderate,3: severe).HumanAATProteinandrAAV8-CB-AATVector AdministrationForhAATproteintherapystudies,DBA/1micewere intraperitoneally(IP)injectedwith0.5mg(in100 lsaline)ofhAAT(Prolastin,BayerCorp.,Elkhard,IN). Thecontrolgroupreceivedsalineinjection.Theinjectionswereperformedtwiceperweek,startingat6days beforethefirstbCIIimmunizationuntiltheendof study(EOS)atday70afterthefirstimmunization.For hAATgenetherapystudies,DBA/1micewereIP injectedwithrAAV8-CB-hAATvector(21011particles/mouse)twoweeksbefor ethefirstCIIimmunization.Thecontrolgroupreceivedsalineinjection.ELISAfortheDetectionofSerumhAATandBAFFLevels andAntibodiesagainsthAAT,bCIIandmCIIDetectionofhAATandanti-hAATantibodiesinmouse serumwasperformedaspreviouslydescribed[32].PurifiedhAAT(AthensResearch&Technology,Athens, GA)wasusedasastandard.Anti-typeIIcollagenantibodiesinmouseserumweredetectedbyastandard ELISA.Briefly,microtiterplates(Immulon4,Dynex Technologies,Chantilly,VA)werecoatedwithbCIIor mCII(0.5 g/well,ChondrexLLC,Redmond,WA)in Voller sbufferovernightat4C.Afterblockingwith3% bovineserumalbumin,wellswereincubatedwithsamplesatroomtemperaturefor2h.HRP-conjugatedgoat anti-mouseIgGantibodies(1:1,000dilution,Sigma, St.Louis,MO),goatanti-mouseIgG1antibodies (1:1,500dilution,Roche,Indianapolis,IN)andgoatantimouseIgG2aantibodies(1:1,500dilution,Roche,Indianapolis,IN)wereincubatedfor1hatRT.Theplates werewashedwithPBS-Tween20betweenreactions. Afteraddingthesubstrate(o-phenylenediamine,Sigma, StLouis,MO),plateswe rereadat490nmonanMRX microplatereader(DynexTechnologies,Chantilly,VA). Opticaldensitieswereconvertedintounitsbasedona standardcurvegeneratedwithhightiterserafrom DBA/1miceimmunizedwithbCII.DetectionofBAFF inserumwasperformedaccordingtomanufactures instructions(R&Dsystems,Inc.Minneapolis,MN).CellCultureThemurinemacrophagecelllineRAW264.7wasculturedinserumfreeDMEMat37Cina5%CO2incubator.FormeasuringBAFFreleaseintomedium,cells wereseededat1105/mlin12wellplates.Cellswere incubatedinquadruplicateswithhAAT(0.5mg/ml;Prolastin,BayerCorp.,Elkhard,IN)for16hoursand BAFFsecretionintotheculturemediumwasdeterminedbyELISAaccordingtomanufacturesinstructions (R&Dsystems,Inc.Minneapolis,MN).QuantitativePCRTotalRNAfromcellculturedescribedabove,wasisolatedusingRNeasyMiniKit(Quiagen,Valencia,CA). Sampleswereprocessedaccordingtothemanufacture s protocol.Forreversetrans cription,cDNAwassynthesizedwitholigodT16primersandMoloneyMurineLeukemiaVirusReverseTranscriptase(MMLV-RT) accordingtomanufacture smanual(TaqmanReverse TranscriptionReagents,AppliedBiosystems,FosterCity, CA). cDNAwasanalyzedbyquantitativePCRusinggenespecificprimerswithSYBRGreen2XPCRmix(Applied Biosystems).Thesequenceoftheprimerswereasfollows:BAFF(205bp),sense:5 -TGCCTTGGAGGA GAAAGAGA-3 andantisense:5 -GGAATTGTT GGGCAGTGTTT-3 ;GAPDH(122bp),sense:5 -CCT GGAGAAACCTGCCAAGTAT-3 andantisense: 5 -TGCTGTTGAAGTCGCAGGA-3 .Reactions weresetupintriplicateandperformedontheABI Prism7700SequenceDetector(AppliedBiosystems). Thecyclingparameterswere2minat95Cfordenaturation,40cyclesof15sat95Cand30sat60Cfor amplification.Thethresholdcycle(CT)ofeachtarget productwasdetermined,settotheloglinearrangeof theamplificationcurveandkeptconstantforalldata analysis.DatawereanalyzedwithSequenceDetector Software(SDS).BAFFexpressionwasnormalizedtothe correspondingGAPDHvaluesfortherespectivetreatment.ValuesofBAFFexpressionfollowingsalinetreatmentaredesignatedas1.Theexperimentwasrepeated twice.AssessmentofT-cellAutoreactiveResponseTotesttheeffectofAAV8-hAATgenetherapyonsplenocyteproliferation,spleen swereharvestedat30days afterthefirstbCIIimmunization.SplenocyteswereisolatedandculturedinserumfreeX-VIVOmediumGrimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page3of13

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(Cambrex,Walkersville,MD)inthepresenceorabsence ofbCII(100 g/ml,ChondrexLLC,Redmond,WA). After3daysculture,1 Ci/wellof[3H]TdRwasadded. Cellswereculturedforadditional18hand[3H]TdR uptakewasmeasuredusinga b -scintillationcounter. Tomeasurecytokinereleaseintothecellculture supernatant,aBeadlyteMouseMulti-CytokineDetectionSystem1kit(Upstate,Temecula,CA,Cat# 48-005)wasusedaccordingtothemanufacture s instructionandinconjunctionwiththeLuminex100 systemforcytokinedetermination.StatisticalAnalysisDataAnalysiswasperformedusingGraphPadPrism4.0 (GraphPadSoftware)andSAS(SASInstitute).Student s t-testwasusedtocomparedifferencesinBAFFlevelsin culturemediumaswellasdifferencesinmRNAexpressionlevels.Mann-WhitneyU-testwasappliedtoanalyze differencesinstimulationindices,cytokinelevels,pathohistologicalchanges,serumlevelsofBAFFandantibodies.Forcomparisonofarthritisscore,areaunderthe curveanalysiswasusedanddifferencesinarthritisincidenceweredeterminedusingKaplan-Meiersurvival curveandlog-ranktest.Ap-valueofp 0.05wasconsideredstatisticallysignificant.ResultsHumanAATProteinTherapyDelayedArthritis DevelopmentinDBA/1MiceInordertoinvestigatetheeffectofhAATondevelopmentofarthritis,wefirstexaminedthefeasibilityof hAATproteintherapyinCIAmousemodel.AdministrationofhAAT(0.5mg/mousetwiceperweek,startingat 6daysbeforetheinductionofarthritis)resultedinsustainedhighlevelsofhAATinmouseserum(Figure1A). Althoughanti-hAAT-antibodiesweredetected(Figure 1B),serumlevelsofhAATdidnotdecreaseovertime. AfewdaysafterthesecondimmunizationwithbCII (day21),miceincontrolgroupdevelopedarthritisinmultiplejoints,whichwasmanifestedbyredness,severejoint swellingandjointstiffnessaswellasankylosisasthediseaseprogressed.Theseverityofarthritisasmeasuredby thearthriticscorerapidlyincreasedincontrolgroup(n= 7)whereasthediseasedevelopmentinhAATtreatment group(n=9)wassuppressed(Figure1C).Atday49 (7weeks)aftertheimmunization,areaunderthecurve (AUC)inthehAATgroupwas50.8321.64(mean SEM),whileincontrolgroupitwas121.517.67(p= 0.029,meanSEM,AUCanalysisuntilday49).Human AATproteintherapyalsoreducedincidenceofsevere arthritis(p=0.0025,logranktest,Figure1D).Moreover, miceinhAATtreatedgrouphadsignificantlydelayed onsetofarthritiscomparedwithcontrolgroup.Onaverage,theclinicalsignsofseverearthritis(arthritisscore >3)startedonday47.38.7(meanSD)inhAATtreatedgroupcomparedtoday36.05.8(meanSD)incontrolgroup(p=0.01bystudentst-test).AlthoughhAAT treatedmicealsodevelopedarthritisattheend(70days aftertheimmunization)oftheexperiment,theseresults showedthattreatmentofhAATprotein(Prolastin)led toadelayedarthritisonsetandameliorationofdisease progressioninCIAmousemodel.HumanAATProteinTherapyReducedtheLevelsofantibCIIandanti-mCIIAutoantibodiesIthasbeenshownthathighlevelsofserumanticollagenIIautoantibodiesarepathognomonicandassociatedwiththedevelopmentofarthritis[36,37].Totest theeffectofhAATonautoantibodyproduction,we evaluatedthelevelsofanti-CIIautoantibodiesintotal Ig,andIgG1andIgG2asubclassatearly(day35)and late(day49)stagesofthedisease.AsshowninFigure 2A,hAATtreatmentdidnotresultinasignificant changeoftotalautoantibodylevelsagainstbCII(total anti-bCII-Ig).However,hAATtreatmentsignificantly reducedthepathognomonicIgG2a(anti-bCII-IgG2a) levelsatday35(Figure2B),andincreasedIgG1(antibCII-IgG1)levelsatday49(Figure2C).Interestingly, levelsoftotalIgautoantibodiesagainstendogenous mousecollagenII(totalanti-mCII-Ig)weresignificantly lowerinhAATproteintreatedgroupthanthoseincontrolgroup(P<0.05)(Figure2D).HumanAAT(hAAT)GeneTherapydelayedArthritis DevelopmentTofurtherconfirmourobservationthathAATiseffectiveindelayingarthritisdevelopment,andtotestthefeasibilityofhAATgenetherapyforrheumatoidarthritis, weusedrecombinantadeno-associatedvirusvector (rAAV)todeliverthehAATgene.AsingleIPinjection ofrAAV8-CB-hAATvector(2x1011particles/mouse,two weeksbeforethefirstCIIimmunization)resultedinsustainedlevelsofhAATinthecirculation,similartothose levelsobtainedfollowingproteintherapy(Figure3A). Interestingly,followingAAV8mediatedgenedelivery,we didnotobservethedevelopmentofantibodiestohAAT whichweredetectedduringhAATproteintherapy(Figure3B,comparevs.Figure1BinmicewithhAATproteintherapy).SimilartotheresultsfromhAATprotein therapy,however,rAAV-m ediatedhAATgenetherapy significantlyreducedtheprevalenceofarthritisdevelopmentattheearlystageofdisease(Figure3C).Area underthecurve(AUC)inthegenetherapygroup(n= 10)was71.6514.04(meanSEM),whileincontrol group(n=10)itwas123.2019.83(meanSEM;p< 0.05byAUCanalysisuntilday42).AATgenetherapy alsoreducedtheincidenceofseverearthritis(score>3) attheearlystageofdisease(p=0.035bylogranktest,Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page4of13

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Figure3D).Moreover,miceinhAATgenetherapygroup hadsignificantlydelayedonsetofarthritiscomparedwith controlgroup.Onaverage,theclinicalsignsofsevere arthritisstartedonday42.37.5(meanSD)inhAAT genetherapygroupcomparedtoday33.47.3incontrolgroup(meanSD;p<0.02bystudent st-test). TheseresultsindicatethatsimilartohAATproteintherapy,AAV8mediatedhAATgenedeliveryalsodelayed arthritisonsetandamelioratedearlystagediseaseprogressioninCIAmousemodel. InanadditionalexperimentusingAAV8mediated hAATgenetherapy,tissueprotectivepropertiesofhAAT wereevaluated.Similartothepreviousexperiment,mice intreatmentgroup(n=6)showedsignificantlyreduced arthritisdevelopmentattheearlydiseasestagecompared tocontrol(n=4)(Figure4A,p<0.05byMann-Whitney U-test).AsshowninFigure4B-F,AAV8mediatedhAAT genetherapyresultedinlessinfiltrationofimmunecells intothejointcavityaccompaniedwithreducedsynovial cellhyperplasiaandpannusformation(p<0.05MannWhitneyU-test).HumanAAT(hAAT)GeneTherapyReducedtheLevelsof Anti-CIIAutoantibodiesAsshowninFigure5,rAAV8-mediatedhAATgene therapyresultedinasignific antsuppressionofanti-CII Figure1 Antiarthriticeffectofhumanalpha1antitrypsin(hAAT)incollageninducedarthritis(CIA)model .HumanAAT(Prolastin)was intraperitoneallyinjectedinDBA/1mice(n=9),twiceperweekstarting6daysbeforeuntilday70afterCIIimmunization.Controlgroup receivedsalineinjections(n=7)( A) SerumhAATproteinlevelsinDBA/1miceweremeasuredbyELISA(mean+SD). indicatesthedayoffirst hAATinjection. (B) Serumanti-hAATantibodylevels(anti-hAAT-IgG)inDBA/1miceweremeasuredbyELISA.Eachdotrepresentsantibodylevels (day49afterbCIIimmunization,arbitraryunits)ofanindividualmouse. (C) Arthritisscore.Foreachpaw,0isnormaland4isthemostsevere arthritis.Themaximumscoreforeachanimalis16.EachlinerepresentsthescoresfromhAATtreatedgroup(opentriangles,mean-SD)or controlgroup(opencircles,mean+SD,*p=0.029byAUCanalysis) (D) Incidenceofseverearthritisisdefinedbyarthriticscore/mouse>3 (**p=0.0025bylogranktest).Dottedline,salineinjectedcontrolgroup;Solidline,hAATtreatedgroup. Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page5of13

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autoantibodyproduction.ThelevelsoftotalIganti-bCII (Figure5A,topleftpanel)andIgG2aanti-bCII(Figure 5A,toprightpanel)weresignificantlyreducedinhAAT genetherapygroup.AlthoughIgG1anti-bCIIlevels (Figure5A,bottomleftpanel)werealsoreducedin hAATgenetherapygroup,theratioofIgG2aanti-bCII toIgG1anti-bCII(Figure5A,bottomrightpanel)significantlydecreasedinhAATgenetherapygroup.Importantly,hAATgenetherapyalsoreducedlevelsof autoantibodiesagainstmCIIandtheratioofIgG2aantimCIItoIgG1anti-mCII(Figure5B).HumanAATTherapyReducedB-cellActivatingFactor (BAFF) invitro and invivoInordertofurtherelucidatetheunderlyingmechanism oftheanti-arthriticeffectofhAAT,weperformedadditionalstudiesfocusingontheeffectofAATonT-cell andB-cellactivity.SinceCIAisaT-cell-mediatedautoimmunedisease,theeffectofhAATonT-cellfunction wasexaminedinaT-cellproliferationassay.Asshown inFigure6A,treatmentofrAAV8-hAATdidnotchange theantigenspecificT-cellresponseafterisolatedsplenocyteswererestimulated exvivo withbCII.Similarly, bCIIinducedcytokinerelease(IFNg ,IL-4,IL-10,TNFa ,IL-2)fromisolatedsplenocytesdidnotshowanysignificantdifferencesbetweentreatmentandcontrol group(Figure6B).TheeffectofhAATtherapyonB-cell activitywasexaminedbydeterminationofserumlevels ofB-cellactivatingfactoroftheTNFa family(BAFF), whichhasemergedasacrucialfactorforB-cellexpansionandfunction.Interestingly,bothhAATproteinas wellasAAV8mediatedhAATgenetherapyresultedin significantlydecreasedserumlevelsofBAFFcompared tocontrolgroup(Figure6C,6D).SinceBAFFismainly Figure2 Anti-collagenII(CII)antibodylevelsafterhAATtreatment .Anti-CIIantibodiesatday35andday49weretestedbyELISA.Closed barsrepresenttheaveragelevels(n=9,relativeunits,mean+SD)ofantibodiesinhAATproteintherapytreatedgroup.Openbarsrepresentthe averagelevels(n=7,relativeunits,mean+SD)ofantibodiesinsalineinjectedgroup. (A) LevelsoftotalIgantibodiestobCII(totalanti-bCII-Ig). (B) LevelsofIgG2aanti-bCII(anti-bCII-IgG2a). (C) LevelsofIgG1anti-bCII(anti-bCII-IgG1). (D) LevelsoftotalIgantibodiestomCII(totalanti-mCIIIg).*p<0.05byMann-WhitneyU-test. Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page6of13

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secretedfrommonocytesandmacrophages,wetested theeffectofhAATonBAFFproduction invitro .Murinemacrophages(RAW264.7)weretreatedwithhAAT. Culturemediumservedascontrol.Proteinsecretion intotheculturemediumwasdeterminedbyELISAand mRNAexpressionwasquantifiedbyreal-timePCR.As showninFigure6E,BAFFlevelsinculturemedium weresignificantlylowerintheAATtreatedgroupthan thoseinthecontrolgroup.Similarly,mRNAexpression levelsofBAFFwerealsosignificantlydecreasedinAAT treatedgroup(Figure6F).Togethertheseresultssuggest thattheanti-arthriticeffectofAATisinpartthrough theinhibitionofB-cellactivation.DiscussionRAisacomplexsystemicautoimmunedisease ofunknownetiology.Althoughrecentlydeveloped biologicsthattargetTNF-alphahaveprovideddramatic improvementincontrollingdiseaseactivityinmany patients,continuedsearchesformoreefficientandsafer treatmentsarestillneeded.Inthepresentstudywe showedthathAAT,administeredasproteinorthrough rAAV8mediatedgenetherapy,reducedlevelsofserum anti-CIIauto-antibodiesandB-cellactivatingfactor (BAFF)andsignificantlydelayedarthritisdevelopmentin amousemodel. Althoughtheexactmechanismsunderlyingthetherapeuticeffectremaintobefurtherinvestigated,several mechanismsmaybeinvolved.Oneisthroughtheinhibitionofproinflammatorycytokineproduction.Itiswell knownthatvariousproinfla mmatorycytokines,includingTNFa andIL1b ,playmajorrolesinthepathogenesisofRA[3].Strategiestargetingthesecytokineshave proventobeeffectiveintreatmentofRA[38].Previous workdonebyJanciauskieneandhercolleaguesclearly demonstratedthathAATinhibitedLPS-inducedTNFa Figure3 HumanAATgenetherapydelaysdiseaseprogressioninCIAmousemodel .DBA/1micewereintraperitoneallyinjectedwith rAAV8-CB-hAATvector(21011particles/mouse,n=10)orsaline(n=10)twoweeksbeforeimmunizationwithCII.Controlgroupreceived saline.Miceweresacrificedonday56(EOS). (A) SerumlevelsofhAAT.hAATproteinserumlevelsinvectorinjectedgroupweremeasuredby ELISA(mean+SD). indicatesthedaysofinjection. (B) Anti-hAATantibodylevels.Serumanti-hAATantibodies(anti-hAAT)weremeasuredby ELISAusingsamplesobtainedat56daysafterimmunization.Anti-hAATantibodieswereundetectableinthevectorinjectedgroup.Eachdot representsantibodylevel(arbitraryunits)ofanindividualmouse. (C) Arthritisscore.EachlinerepresentstheaveragescorefromhAATtreated group(opentriangles,mean-SD)orcontrolgroup(opencircles,mean+SD,*p<0.05asdeterminedbyAUCanalysis.) (D) Incidenceofsevere arthritis.Severearthritiswasdefinedbyarthriticscore>3,(*p=0.035bylogranktest.;10mice/group). Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page7of13

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Figure4 TissueprotectiveeffectofhAATgenetherapyinCIAmousemodel .DBA/1micewereintraperitoneallyinjectedwithrAAV8-CBhAATvector(21011particles/mouse,n=6)orsaline(n=4)twoweeksbeforeimmunizationwithCII.Controlgroupreceivedsaline. (A) Arthritisdevelopmentwasevaluatedbasedonarthritisscore(mean+SD).OpencirclerepresentrAAV8-CB-hAATvectorinjectedgroup,open trianglerepresentcontrolgroup.Miceweresacrificedonday28afterCIIimmunization,hindlimbswereharvestedandprocessedforhistological assessment.*p<0.05byMann-WhitneyU-test. (B) Histopathologicalevaluationofarthritisdevelopment.Miceingenetherapygroup(black bars)orcontrolgroup(emptybars)wereevaluatedaccordingtohistopathologicalchangesbytwoblindedpathologists.Eachhindpawwas evaluatedbasedonascalerangingfrom0-3.(mean+SD).*p<0.05,**p<0.01byMann-WhitneyU-test.(INF:InfiltrationofImmuneCells,HYP: Hyperplasia,P.F.:PannusFormation,B.D.:BoneDestruction)( C,D) RepresentativejointsectionfrommicereceivinghAATgenetherapy. (E,F) Representativejointsectionfrommiceincontrolgroup(salineinjection).Magnification:C,E:100x;D,F:200x. Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page8of13

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Figure5 EffectofhAATgenetherapyonauto-antibodyproduction .Anti-CIIantibodiesatday28,42and56weretestedbyELISA.Black barsrepresenttheaveragelevels(n=10,mean+SD)(relativeunits)ofantibodiesinhAATgenetherapytreatedgroup.Openbarsrepresentthe averagelevels(n=10,relativeunits,mean+SD)ofantibodiesinsalineinjectedgroup. (A) AntibodylevelsagainstbovineCII(bCII). Topleftpanel totalIgantibodiesagainstbCII(totalanti-bCII-Ig); Toprightpanel ,levelsofIgG2aanti-bCII(anti-bCII-IgG2a); Bottomleftpanel ,levelsofIgG1antibCII(anti-bCII-IgG1); Bottomrightpanel ,theratioofanti-bCII-IgG2atoanti-bCII-IgG1(anti-bCII-IgG2a/IgG1ratio). (B) Antibodylevelsagainst mouseCII(mCII). Topleftpanel ,totalIgantibodiesagainstmCII(totalanti-mCII-Ig); Toprightpanel ,levelsofIgG2aanti-mCII(anti-mCII-IgG2a); Bottomleftpanel ,levelsofIgG1anti-mCII(anti-mCII-IgG1); Bottomrightpanel ,theratioofanti-mCII-IgG2atoanti-mCII-IgG1(anti-mCII-IgG2a/ IgG1).*p<0.05,**p<0.01,***p<0.001byMann-WhitneyU-test. Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page9of13

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Figure6 EffectsofhAATtherapyonT-cellsandB-cells (A) Proliferativeresponseofsplenocytes afterstimulationwithbovinetypeII collagen(bCII,10 g/ml).Splenocytes(4105cells/well,in96-wellplate)wereisolatedonday28afterrAAV8-hAATinjection.Blackbar,AAT genetherapygroup(n=6);openbar,controlgroup(n=4).Dataareexpressedasthestimulationindex,determinedbycalculatingtheratioof cellproliferationwithantigen(measuredincountsperminute,cpm)relativetothatwithmediumalone(mean+SD). (B) Cytokineproduction frombCII-stimulated(100 g/ml)splenocytes.Valuesarethemean+SDofeachgroup(n=6forrAAV8-hAATgroup,blackbars;n=4forsaline group,openbars). (C) SerumlevelofBAFFinhAATtreatedmice(blackbar,n=9,day35)andcontrolmice(openbar,n=7).Dataisexpressed asmean+SD. (D) BAFFserumlevelinrAAV8-hAATtreatedmice(blackbar,n=10,day28)andcontrol(openbar,n=10). Invitro effectofhAAT on (E) BAFFsecretionintoculturemediummeasuredbyELISAand (F) BAFFgeneexpressiondeterminedbyreal-timePCR.Murinemacrophages (RAW264.7)weretreatedwithhAAT(0.5mg/ml,blackbar).Culturemediumservedascontrol(openbar).Bothexperimentswereperformedin quadruplicatesandrepeatedtwice.Dataisexpressedasmean+SD.*p<0.05,**p<0.01. Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page10of13

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IL-6andIL-1 b productionbyhumanmonocytes[15,16]. Inaddition,hAATcompletelysuppressedmacrophage inflammatoryprotein-2(MIP-2)/monocytechemotactic protein-1(MCP-1)geneexpressioninlung[39].Human AATalsoenhancedanti-inflammatorycytokineIL-10 productionfrommonocytes[15].Asaconsequenceof interferingwiththecytokine/chemokinenetwork,hAAT mayalsoinhibitpolymorpho nuclearleukocyte(PMN) invasionintothejoint.Churgetal.demonstratedthat hAATinhibitedsilica-inducedPMNinfluxintothelung andpartiallysuppressednucleartranscriptionfactorB (NFB)translocationandincreasedinhibitorofNFB (IB)levelsinamousemodelofacutePMNmediated inflammation[39].Thus,itispossiblethattheeffects ofhAATonpro-inflammato rycytokineproduction contributetosuppressionofautoimmune-mediated inflammation. InpreviousstudiesweshowedthathAATreduced anti-insulinauto-antibodies(IAA)andattenuatedcellmediatedautoimmunity[32,33].Consistentwiththese results,thepresentstud yshowedthathAATreduced thelevelsofanti-CIIauto-antibodiesandtheIgG2a/ IgG1ratiosofanti-CIIauto-antibodies(mCIIandbCII). WehaveobservedthattheeffectofhAATtosuppress arthritisdevelopmentismoreprofoundinearlystageof arthritisdevelopment.Thisissupportedbytheeffectof hAATonpathognomonicIgG2aantibodydevelopment atearlytimepoints(Fig.2)aswellastheobservation thatmiceeventuallydeveloparthritisovertime.Therefore,hAATmaybeespeciallysuitableforcombination therapies.WedidnotobservesignificanteffectofAAT onT-cellproliferationandcytokineproduction invitro (Figure6Aand6B)indicatingthatAATmayhavelimiteddirecteffectonT-cells.Thesedataalsosuggestthat AATmaymoredirectlyaffectB-cellactivity.Indeed,we haveshownthatAATtherapiessignificantlyreduced B-cellactivatingfactoroftheTNFa family(BAFF) invitro and invivo .BAFFisanimportantfactorthat modulatesB-celltoleranceandhomeostasis.Ithasbeen shownthatsolubleBAFFiselevatedinserumandtarget organsofCIAmodel[40]andBAFFantagonistssuppressedarthritisdevelopmentinmurinemodelsofrheumatoidarthritis[41].Inaddition,increasedBAFFlevels werefoundinserumofRApatientswhichcorrelated withserumlevelsofrheumatoidfactor[42].Theexact mechanismthatAATsuppressesBAFFproduction remainstobeelucidated. AnotherpossiblemechanismofhAATsuppressing arthritisdevelopmentisthroughinhibitionofproteinasestopreventtissueinjuryandjointdestruction. HumanAATiswellknownasaserineproteinaseinhibitor(serpin).Itinhibitspr oteinase3,neutrophilelastase,andcathepsinG.Theseserineproteasesare releasedbyjointinvadingneutrophilsfollowing inflammatorystimuliandhaveshowntobeinvolvedin arthritisdevelopment[ 12,13,43,44].HumanAATcan alsoreduceischemia-induced apoptosis,inflammation, andacutephaseresponseinthekidney[20].Wehave recentlyshownthathAATdi rectlyinhibitscaspase3 activityandprotectsisletcellsfromcytokineandchemically-inducedapoptosis[45]. Intheproteintherapystudies,weusedProlastin, whichisclinicalgradehAATpurifiedfromhuman plasma.RepeatedIPinjectionofhAATinducedstrong humoralimmuneresponseagainsthAATinDBA/1 mice(Figure1B),similartowhathasbeenobservedin previousstudies[46,47].Itispossiblethatnon-specific inflammationcausedbyrepeatedIPinjectionisresponsibleforinhibitionofarthritis.Inordertoruleoutthis possibility,weperformedrAAV8mediatedhAATgene therapy.AAVserotype8vectorisuniqueforthispurposebecauseitcanmediatelongtermandhighlevels oftransgeneexpressionintheliverandmuscle,butis notabletotransducedendriticcellsandhaslowimmunogenicity[48,49].Indeed ,afterasingleinjectionof rAAV8-CB-hAATvector,sustainedhighlevelsofhAAT weredetectedinthecircula tion,whilenodetectable levelsofanti-hAATantibodieswerepresent(Figure3B) incontrasttomicethatreceivedhAATproteintherapy. TheseresultsareconsistentwithourrecentobservationsinNODmiceandimplynewapplicationsof rAAV8vectors[34].Thedetailedmechanismthat rAAV8vectormediatesnoimmuneresponsetothe transgeneproductremainselusive.Importantly,wehave observedprotectiveeffectsandreductionsofautoantibodiesbyhAATgenetherapy.Theseresultsstrongly supportourhypothesisth athAATisabletoreduce inflammationinautoimmunediseases,suchasRAand type1diabetes.ConclusionOurresultsfromproteinandgenetherapyshowedthat hAATiseffectiveindelayingarthritisdevelopmentina mousemodelofCIA.TheyindicatethathAAThas immunoregulatoryandimm unomodulatoryeffectsand hasgreatpotentialasanewtreatmentforRA.Wealso haveshownthatrAAV8mediatedgenetherapyresulted inareducedimmuneresponsetothetransgeneproduct. Futurestudieswillfocusonimprovementofthetherapeuticeffectbyoptimizingthedoseandtimingof hAATorrAAV8vectordelivery,andbycombination therapywithotheranti-arthriticdrugs.Abbreviations hAAT:humanAlpha-1Antitrypsin;CIA:CollagenInducedArthritis;IFA: IncompleteFreund sAdjuvant;CFA:CompleteFreund sAdjuvant;RA: RheumatoidArthritis;NOD:NonObeseDiabetic;bCII:bovinetypeII Collagen;mCII:mousetypeIICollagen;TNF:TumorNecrosisFactor-alpha;Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page11of13

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IL:Interleukin;LPS:Lipopolysaccharide;PBMC:PeripheralBloodMononuclear Cells;BAFF:B-cellActivationFactoroftheTNFFamily;rAAV:Recombinant Adeno-AssociatedVirus;MMP:Matrix-Metalloproteinase;ELISA:EnzymeLinkedImmunosorbentAssay Acknowledgements ThisworkwassupportedbygrantsfromNIH(DK58327)andUniversityof FloridaOfficeofResearch. Authordetails1DepartmentofPharmaceutics,UniversityofFlorida,Gainesville,FL32610, USA.2DepartmentofPathology,UniversityofFlorida,Gainesville,FL32610, USA.3DepartmentofMedicine,UniversityofFlorida,Gainesville,FL32610, USA. Authors contributions CGconceivedofthestudy,participatedinitsdesign,carriedoutanimal experiments,cellproliferationassay,immunoassays,performedstatistical analysisanddraftedthemanuscript.YKCconceivedofthestudy, participatedinitsdesignandperformedanimalexperimentsandcell proliferationassay.CWhelpedperformingcellproliferationassay,MS participatedindiscussionandhelpedtorevisethemanuscript,MA,MCT andMBparticipatedindesignanddiscussionofthestudy,SSconceivedof thestudyparticipatedinitsdesignandhelpedtorevisethemanuscript.All authorsreadandapprovedthefinalmanuscript. Competinginterests ChristianGrimsteinandSihongSongmaybeentitledtofuturepatent royaltiesfromtechnologydescribedinthispaper. Received:4October2010Accepted:24February2011 Published:24February2011 References1.FiresteinGS: Evolvingconceptsofrheumatoidarthritis. Nature 2003, 423 :356-361. 2.EdwardsCJ,CooperC: Earlyenvironmentalfactorsandrheumatoid arthritis. 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GeneTher 2001, 8 :1248-1254. 32.SongS,GoudyK,Campbell-ThompsonM,WasserfallC,Scott-JorgensenM, WangJ,TangQ,CrawfordJM,EllisTM,AtkinsonMA,FlotteTR: Recombinantadeno-associatedvirus-mediatedalpha-1antitrypsingene therapypreventstypeIdiabetesinNODmice. GeneTher 2004, 11 :181-186. 33.LuY,TangM,WasserfallC,KouZ,Campbell-ThompsonM,GardemannT, CrawfordJ,AtkinsonM,SongS: Alpha1-antitrypsingenetherapy modulatescellularimmunityandefficientlypreventstype1diabetesin nonobesediabeticmice. HumGeneTher 2006, 17 :625-634.Grimstein etal JournalofTranslationalMedicine 2011, 9 :21 http://www.translational-medicine.com/content/9/1/21 Page12of13

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