novel stable cell lines for constitutive lentiviral vector production

Abstract. Lentiviral vectors (LVs) are excellent tools to promote gene transfer and stable gene expression. Their potential has been already ... Skiptomaincontent Thankyouforvisitingnature.com.YouareusingabrowserversionwithlimitedsupportforCSS.Toobtain thebestexperience,werecommendyouuseamoreuptodatebrowser(orturnoffcompatibilitymodein InternetExplorer).Inthemeantime,toensurecontinuedsupport,wearedisplayingthesitewithoutstyles andJavaScript. Advertisement nature scientificreports articles article LentiPro26:novelstablecelllinesforconstitutivelentiviralvectorproduction DownloadPDF DownloadPDF Subjects GenetherapyGeneticvectorsRetrovirus AbstractLentiviralvectors(LVs)areexcellenttoolstopromotegenetransferandstablegeneexpression.Theirpotentialhasbeenalreadydemonstratedingenetherapyclinicaltrialsforthetreatmentofdiversedisorders.ForlargescaleLVproduction,astableproducersystemisdesirablesinceitallowsscalableandcost-effectiveviralproductions,withincreasedreproducibilityandsafety.However,thedevelopmentofstablesystemshasbeenchallengingandtime-consuming,beingtheselectionofcellspresentinghighexpressionlevelsofGag-Pro-Polpolyproteinandthecytotoxicityassociatedwithsomeviralcomponents,themainlimitations.HerebyisdescribedtheestablishmentofanewLVproducercelllineusingamutatedlessactiveviralproteasetoovercomepotentialcytotoxiclimitations.Thestabletransfectionofbicistronicexpressioncassetteswithre-initiationofthetranslationmechanismenabledthegenerationofLentiPro26packagingpopulationssupportinghightiters.Additionally,byskippingintermediateclonescreeningstepsandperformingonlyonefinalclonescreening,itwaspossibletosavetimeandgenerateLentiPro26-A59cellline,thatconstitutivelyproducestitersabove106TU.mL−1.day−1,inlessthansixmonths.ThisworkconstitutesastepforwardtowardsthedevelopmentofimprovedLVproducercelllines,aimingtoefficientlysupplytheclinicalexpandinggenetherapyapplications. IntroductionLentiviralvectors(LVs)areremarkabletoolsforgenetransfer,bothinvivoandinvitro,duetotheirabilitytopermanentlyintegrateintothecellgenomeofdividingandnon-dividingcells,sustaininglong-termstableexpression.SincethefirstsLVdevelopmentsintheearly1990s,LVshavebeenimprovedintermsofsafetyandefficacy1;noteworthyisthedevelopmentof3rdgenerationpackagingsystem2andself-inactivating(SIN)vectors3,4.LVshavealsobeenreportedtoexhibitlowergenotoxicitywhencomparedtotheirsimplercounterparts,γ-retroviralvectors(γ-RVs)5,6.ThesereasonsjustifythegrowingnumberofLVbasedgenetherapystudiesenteringclinicaltrials7,withpromisingresultsforthetreatmentofneurodegenerativeorgeneticdisorders,infectiousdiseases,andmorerecentlycancerimmunotherapy8,9.Veryrecently,theUnitedStatesFoodandDrugAdministrationapprovedthefirstgenetherapytreatmentusingLVs,thetisagenlecleucel(marketedasKYMRIAH™).ThistreatmentusesaSIN-LVtomodifyautologousTcellsandtreatBcellacutelymphoblasticleukaemia.Theapprovaloftisagenlecleucelmarkstheclinical-to-markettransitionofLVsintothegenetherapyfield,anticipatingtheneedforlarge-scaleproductionofclinicalgradeLVpreparations.Traditionally,theproductionofLVshasreliedontransientco-transfectionofHEK293Tcellswithfourexpressioncassettes:(i)Gag-Pro-Pol,codingfortheviralstructuralproteinsandenzymes;(ii)Rev,encodingRevaccessoryproteinwhichplaysanimportantroleonviralgenomenuclearexportation;(iii)Envelope,codingfortheglycoproteinsthatinteractwithtargetcellreceptorstomediateviruscellentry;and(iv)Vectorgenome,carryingthegeneofinteresttobepackagedintoproducedviralparticles.TransientLVproductionsareeasytoperformatsmallscale.However,transientLVproductionsaredifficulttoscale-up.AdditionaldisadvantagesincludetheheavycostsofhighqualitytransfectableDNAandtransfectionreagents,substantialbatch-to-batchvariabilityandshortproductionperiods10.Inthiscontext,astableLVcelllineconstitutivelyproducinghightiterLVsishighlydesirable.However,thegenerationofsuchcelllinesischallengingandtimeconsuming,requiringseveraltransductions/transfectionsandselectionsteps,allofwhichcombinedwithclonalisolation,amplificationandscreening,tofindthebestLVproducerclones.Inaddition,thecytotoxicityofbothHIV-1proteaseandglycoproteinfromvesicularstomatitisvirus(VSV-G)envelope(themostusedenvelopetopseudotypeLVs)hashamperedtheestablishmentofstableLVproducercelllines1.Todealwiththecytotoxicityissues,celllineswithinduciblesystemsfortheexpressionofviralcomponentshavebeendeveloped10.Yet,whenthoseinduciblecelllinesareused,LVproductionismaintainedonlyforashortperiodoftimeafterinductionandadditionalpurificationstepsmayberequiredtoremovetheinducibleagents10.Todate,onlythreecelllineshavebeenreportedconstitutivelyexpressingalltheLVcomponents(Gag-Pro-Pol,REV,EnvelopeandVectorgenome).Inallthecases,theselectionofaclonewithstableandhighexpressionofGag-Pro-Polseemstobethemajorchallenge,beingdifficulttodevelopastablehightiterLVproducercelllineexclusivelybytraditionalplasmidcelltransfection.Toovercomeexpressionlimitations,severalstrategiesweredevelopedusingviralvectorstointroducetheLVcomponentsintocellgenomes,promoting theirhighexpressionandfacilitatingtheestablishmentofclonesproducinghighLVtiters.TheSTARderivedcelllines11werethefirstLVproducercelllinestobeestablished.Duringitsdevelopment,γ-RVswereusedtointegrateagag-pro-polcodon-optimizedandrevexpressioncassettesintothegenomeofHEK293Tcells.TheremainingLVcomponentswereintroducedbyplasmidcelltransfection.TheSTARestablishmentevidencedthatispossibletodevelopacelllineconstitutivelysupportinghighLVproductivities.However,theLongTerminalRepeats(LTRs)andpackaging(Ψ)sequencesoftheγ-RVsusedinSTARcelllinedevelopmentarepresentinthegenomeoftheLVproducercells,whichcouldpromotethegenerationofreplicationcompetentlentivirus(RCL),raisingsafetyconcerns12.Someyearslater,theRD2-MolPack-Chim3LVproducercelllinewasdevelopedusingarecombinanthybridbaculo-AAVvectortosuccessfullyintegratethegag-pro-polandrevgenesintothecellgenome,avoidingtheusageofγ-RVs13.TheTatandenvelopegeneswereintroducedusingSIN-LVstominimizepossiblesafetyconcerns14,15.PosteriorspecificanalysisattestedthesafetyoftheRD2-MolPack-Chim3cellline13.Morerecently,inWinPacderivedcelllinesdevelopment,adifferentapproachusedγ-RVstointegrateareporterexpressioncassetteintothecellsgenometoidentifyaclonesupportinghighreporterexpressionlevels16.Subsequently,thisreporterexpressioncassettewasreplacedbyanewonecontainingacodon-optimizedLVgag-pro-polsequence,bymeansofCrerecombinasemediatedcassetteexchange(RMCE).TheremainingLVcomponentswereintroducedbythetraditionalplasmidcelltransfection.Theremovalofmostofγ-RVsequencesduringthecassetteexchangeeventdecreasestheriskofRCLformation.Nevertheless,theusageoftheRMCErequiredadditionalstepsofcloneisolationandscreening,makingcelllinedevelopmentevenlongerandmorelaborious.AllthethreeLVproducercelllinesmentionedreflecttheactivedemandforimprovedstableLVproducersystems.Herein,isdescribedanalternativemethodologytoacceleratetheestablishmentofLVproducercelllinespresentinghightiters,exclusivelybyusingchemicaltransfectionsfollowedantibioticselectionstepsduringtheentirecelllinedevelopmentprocess.ResultsTransientLVproductionsusingT26SmutatedorwildtypeviralproteaseTheT26SpointmutationwasperformedintheviralproteaseofpMDLg/pRREplasmid2,originatingthepGP(T26S)P(Fig. 1a).Thismutationwasreportedtodecreaseproteaseactivitywithoutaffectingvirusmaturationandinfectivity17,potentiallyleadingtolowercytotoxicitywhenstablyexpressed.Ultimately,thiscouldsupporthigherexpressionlevelsofGag-Pro(T26S)-Pol.ThefunctionalityofT26SproteasewasassessedbytransientproductionofLVspseudotypedwithVSV-Gorwithamphotropicenvelope(Fig. 2a).Asacontrol,thewildtypeproteasewasalsoevaluated.NodifferencesininfectiousviraltitersobtainedwereobservedforLVproductionswithVSV-Genvelope,whereasa2-folddecreaseoninfectiousLVtiterwasdetectedforviralproductionusingtheT26Smutatedproteasewithamphotropicenvelope.Titersabove107TU.mL−1.day−1wereachievedforallLVproductionsandtheamphotropicenvelopewasusedtoproceedwithstablecelllineestablishment.Figure1Schematicrepresentationoftheexpressioncassettesusedinthiswork.(a)Gag-Pro-Polexpressioncassettes.(b)Revexpressioncassettes.(c)Envelopeexpressioncassettes.(d)LVgenomeexpressioncassettes.Abbreviations:CMV,Cytomegaloviruspromoter;Int,intron;GPP,gag-pro-polsequence;pAn,polyAsequence;GP(T26S)P,gag-pro-polwithmutatedT26Sproteasesequence;BlastR,blasticidinresistancegene;RSV,RousSarcomaViruspromoter;HygroR,hygromycinresistancegene;WPRE,woodchuckhepatitispost-transcriptionalregulatoryelement;VSV-G,glycoproteinGofthevesicularstomatitisvirus;SV40/FerH,ferritinheavychain(FerH)promoterfusedwithSV40enhancer;IRES,internalribosomeentrysite;ZeoR,zeocinresistancegene;Ψ,packagingsignalsequence;hPGK,humanphosphoglyceratekinasepromoter;PuroR,puromycinresistancegene.ThethinnergreyarrowsrepresentthemRNAtranscripts.Thefilledgreyboxesrepresentthespacerregionthatdrivesthere-initiationofthetranslationmechanism.FullsizeimageFigure2TransientLVproductionsbytransfectingHEK293Tcells.(a)TransientproductiontitercomparisonofLVpseudotypedwithVSV-Goramphotropicenvelope,usingthewildtypeorthemutatedT26Sprotease.(b)TransientLVproductiontitercomparisonofGag-Pro(T26S)-Polexpressioncassettes.(c)TransientLVproductiontitercomparisonofRevexpressioncassettes.(d)TransientLVproductiontitercomparisonofvectorgenomeexpressioncassettes.The(+)indicatespresenceandthe(−)indicatesabsenceoftherespectiveplasmidinthetransfectionmix.Datashownrepresentstheaverage ± standarddeviationfrom3independentexperiments.*(p