Long-term immune reconstitution in RAG-1-deficient mice ...

Abstract. Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and ... SkiptoMainContent Advertisement Close ASHClinicalNews ASHNewsDaily ASH-SAP Blood BloodAdvances Hematology TheHematologist International BloodChineseEdition BloodJapaneseEdition BloodItalianEdition BloodLatinAmericaEdition BloodSpanishEdition ASH ASHHome Research Education Advocacy Meetings Publications ASHStore Cart UserToolsDropdown Cart SignIn SearchDropdownMenu navsearch searchinput Searchinputautosuggest searchfilter AllContentAllJournalsBlood Search ToggleMenuMenu Issues CurrentIssue AllIssues Firstedition Abstracts 2021AnnualMeeting 2020AnnualMeeting 2020LateBreaking 2019AnnualMeeting 2019LateBreaking AllMeetingAbstracts Collections Collections SpecialCollections Multimedia Alerts AuthorCenter Submit AuthorGuide StyleGuide WhySubmittoBlood? About AboutBlood EditorialBoardandStaff Subscriptions PublicAccess Copyright Alerts BloodClassifieds SkipNavDestination ContentMenu Close Abstract Introduction Materialsandmethods Results Discussion ArticleNavigation GENETHERAPY| January1,2006 Long-termimmunereconstitutioninRAG-1-deficientmicetreatedbyretroviralgenetherapy:abalancebetweenefficiencyandtoxicity ChantalLagresle-Peyrou, ChantalLagresle-Peyrou FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar FrankYates, FrankYates FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar MichèleMalassis-Séris, MichèleMalassis-Séris FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar ChristopheHue, ChristopheHue FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar EstelleMorillon, EstelleMorillon FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar AlexandrineGarrigue, AlexandrineGarrigue FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar AllenLiu, AllenLiu FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar PhilippeHajdari, PhilippeHajdari FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar DanielStockholm, DanielStockholm FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar OlivierDanos, OlivierDanos FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar BrigitteLemercier, BrigitteLemercier FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar Marie-LiseGougeon, Marie-LiseGougeon FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar FredericRieux-Laucat, FredericRieux-Laucat FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar Jean-PierredeVillartay, Jean-PierredeVillartay FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar AlainFischer, AlainFischer FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar MarinaCavazzana-Calvo MarinaCavazzana-Calvo FromtheUniversitéParis-Descartes,Facultédemédecine,InstitutNationaldelaSantéetdelaRechercheMédicale(INSERM)Unité(U)429,siteNecker-EnfantsMalades,France;DepartmentofBiotherapy,AssistancePublique-HôpitauxdeParis(AP-HP),HôpitalNecker-EnfantsMalades,Paris,France;CentreNationaldelaRechercheScientifique,UniteMixtedeRecherche(UMR)8115,Généthon,Evry,France;Immunitéanti-virale,biothérapieetvaccins,InsermU668,InstitutPasteur,Paris,France;AP-HP,France;andUnitéd'Immunologieetd'HématologiePédiatriques,AP-HP,HôpitalNecker-EnfantsMalades,Paris,France. Searchforotherworksbythisauthoron: ThisSite PubMed GoogleScholar Blood(2006)107(1):63–72. https://doi.org/10.1182/blood-2005-05-2032 Articlehistory Submitted: May20,2005 Accepted: August29,2005 Split-Screen ShareIcon Share Twitter LinkedIn ToolsIcon Tools RequestPermissions CiteIcon Cite SearchSite PDF Citation ChantalLagresle-Peyrou,FrankYates,MichèleMalassis-Séris,ChristopheHue,EstelleMorillon,AlexandrineGarrigue,AllenLiu,PhilippeHajdari,DanielStockholm,OlivierDanos,BrigitteLemercier,Marie-LiseGougeon,FredericRieux-Laucat,Jean-PierredeVillartay,AlainFischer,MarinaCavazzana-Calvo;Long-termimmunereconstitutioninRAG-1-deficientmicetreatedbyretroviralgenetherapy:abalancebetweenefficiencyandtoxicity.Blood2006;107(1):63–72.doi:https://doi.org/10.1182/blood-2005-05-2032 Downloadcitationfile: Ris(Zotero) ReferenceManager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbarsearch SearchDropdownMenu navsearch searchinput Searchinputautosuggest searchfilter AllContentAllJournalsBlood Search Abstract Severecombinedimmunodeficiency(SCID)causedbymutationsinRAG1orRAG2genesischaracterizedbyacompleteblockinT-andB-celldevelopment.Theonlycurativetreatmentisallogeneichematopoieticstemcelltransplantation,whichgivesahighsurvivalrate(90%)whenanHLA-genoidenticaldonorexistsbutunsatisfactoryresultswhenonlypartiallycompatibledonorsareavailable.WehavethusbeeninterestedinthedevelopmentofapotentialalternativetreatmentbyusingretroviralgenetransferofanormalcopyofRAG1cDNA.WeshowherethatthisapproachappliedtoRAG-1-deficientmicerestoresnormalB-andT-cellfunctioneveninthepresenceofareducednumberofmatureBcells.Thereconstitutionisstableovertime,attestingtoaselectiveadvantageoftransducedprogenitors.Notably,ahightransgenecopynumberwasdetectedinalllymphoidorgans,andthiswasassociatedwithariskoflymphoproliferationasobservedinonemouse.Altogether,theseresultsdemonstratethatcorrectionofRAG-1deficiencycanbeachievedbygenetherapyinimmunodeficientmicebutthathumanapplicationwouldrequiretheuseofself-inactivatedvectortodecreasetheriskoflymphoproliferativediseases. Subjects: GeneTherapy, ImmunobiologyandImmunotherapy, Neoplasia Topics: genetherapy, mice, rag1gene, retroviridae, toxiceffect, b-lymphocytes, genetransfertechniques, transgenes, immunereconstitution, immunologicdeficiencysyndromes Introduction Severecombinedimmunodeficiency(SCID)isaheterogeneousgroupofrareautosomicrecessivedisordersoccurringin1in75000to1in100000births.ThecommoncharacteristicofthesepathologiesistheabsenceofTcellsassociatedwithanimpairedfunctionofBlymphocytesleadingtodeathwithinthefirstyearoflifeintheabsenceoftreatment.1,2 Currently,thetreatmentofchoiceisallogeneichematopoieticstemcelltransplantation(HSCT)withasurvivalrateapproximating90%whenanHLA-genoidenticaldonorisavailable.3 Inmostcases,haploidenticaldonorsorunrelateddonorsaretheonlysourceofallogeneichematopoieticstemcells,andinthiscasethesurvivalraterangesfrom60%to75%.WithintheSCIDsubsets,theT-cell-negative,B-cell-negative,naturalkillercell-positive(T-B-NK+)grouphasapoorerprognosis,especiallywhenapartiallycompatibledonorisused(withasurvivalratecloseto36%).Ofnote,thissubgroupofmismatchedpatientsreceivingtransplantspresentsanumberoflong-termcomplicationsduetoafrequentpersistenceofB-celldeficiencyandalong-termdeclineinT-cellfunctionsrelatedtotheabsenceofdonorstemcellengraftment.4 TheslowkineticsoftheT-lymphocytedevelopmentalsoaccountsformanylethalinfectionsobservedinthefirst6monthsafterHSCT.TheselimitationsjustifythesearchforanalternativestrategytoimprovetheprognosisofSCID.TransferofanormalcopyofthemutatedgenethatcausestheSCIDdiseasetoearlyhematopoieticprogenitorshaslargelybeenconsidered,andthistherapeuticapproachhasprovidedefficientlong-termcorrectionin2humanSCIDdiseases5-8 aswellasinvariousimmunodeficientmousemodels.9-12  TheT-B-NK+groupofSCIDdiseaserepresents20%to30%oftheallSCIDforms.Withinthissubset,mutationsin1ofthe2recombinationactivatinggenes,RAG1orRAG2,13,14 accountforhalfofthecases.BothRAG-1andRAG-2proteinsplayakeyroleintheinitiationoftheV(D)Jrecombinationstep,crucialfortheformationoftheantigenreceptoratthesurfaceofTandBlymphocytes.15,16 ThesimultaneousexpressionofRAG-1andRAG-2isrequiredduringtheearlystagesofT-andB-lymphocytematurationand,intheabsenceof1ofthe2proteins,lymphoiddifferentiationisblockedatthepro-Bandtriple-negativepre-Tstages.17 Ofnote,inhumans,hypomorphicmutationsofRAGproteinscanleadtoaleakyphenotype,theOmennsyndrome,characterizedbythepresenceofafewautoreactiveT-cellclones.18 RAG-1-andRAG-2-deficientmice(RAG-1-/-andRAG-2-/-,respectively)exhibitanidenticalphenotype19,20 totheoneobservedinRAG-deficientpatientswithnullmutations.Therefore,theseanimalsrepresentagoodmodeltotesttheefficiencyandtoxicityofRAG1genetherapyapproach. WehavepreviouslydemonstratedthatRAG-2deficiencycouldbelong-termcorrectedfollowingretroviraltransferofthehumanRAG2geneintheHSCsofRAG-2-/-mice.11 Usingthesamestrategy,thehumanRAG1(hRAG1)transgenewasclonedinaMoloneyleukemiavirus(MLV)vectorusedtotransducebonemarrowprogenitorsofRAG-1-/--deficientmice.T-andB-celldifferentiationwasrestoredinallthelymphoidorgansformorethan1yearaftertransplantationprovidedthatthetransgenecopynumberpercellwashighenough.Ofnote,suchahighproviruscopynumberleadstotheoccurrenceofoneleukemicevent.Togetherwiththeobservedclinicalcomplicationsintheγcdeficiencygene-therapytrial21 theseresultsindicatethatalternativetransductionmethods,suchaslong-terminalrepeat(LTR)-inactivatedretroviralvectors,shouldbeusedforthistherapeuticapproach. Materialsandmethods Mice Four-to6-week-oldC57BL/10wild-typemiceandC57BL/10RAG-1-deficientmicewereobtainedfromCharlesRiverLaboratories(L'Arbresle,France).Theanimalsweremaintainedinaspecificpathogen-freeanimalfacility.AllexperimentsandprocedureswereperformedincompliancewiththeFrenchMinistryofAgricultureRegulationsforAnimalExperimentations(Actno.87847,October19,1987,modifiedMay2001). Retroviralsupernatantproduction MFG-RAG1vectorwasobtainedbyaSalI/NcoIligationofthehumanRAG1cDNAproductintotheMFGB2plasmid,whichhasbeendescribedelsewhere.22 RetroviralsupernatantwasproducedinStemspanmedium(StemCellTechnologies,Vancouver,BC,Canada)supplementedwith10%fetalbovineserum(FBS;StemCellTechnologies).Briefly,ecotropicphoenixproducercelllinesweretransfectedfollowingacalcium-phosphateprotocolaspreviouslydescribed.11 After48hours,thesupernatantwascollectedandconcentratedontovivaspinconcentratortubes(Vivascience,Hannover,Germany).Virustitrationwasperformedon3T3celllines,estimatedat6×106infectiousparticlespermilliliterandusedatamultiplicityofinfectionof3.Thedetectionofreplication-competentretrovirus(RCR)intheretroviralsupernatantbatcheswasperformedbyTexcell(Paris,France).23 NoRCRsweredetected. Transductionofbonemarrowcells Bonemarrowcellswereflushedfrombothtibiasandfemoraofdonormiceandtreatedwitha0.75%NH4Cl(Sigma-Aldrich,StLouis,MO)solutioninTris(tris(hydroxymethyl)aminomethanebuffer)(Sigma-Aldrich)toremoveredbloodcells(RBCs).Cellswerestainedwithphycoerythrin(PE)-conjugatedSca-1Ly6A/E(E13-161-7)antibody(BectonDickinson[BD]BiosciencesPharmingen,SanJose,CA),andSca-1+cellswerepurifiedusingananti-PEmagneticselectionkit(MiltenyiBiotec,BergischGladbach,Germany).Sca-1+cellswereculturedfor24hoursinStemspanmedium(StemCellTechnologies)supplementedwith5%FBS(StemCellTechnologies)andthefollowingrecombinantcytokines:murinestemcellfactor(mSCF)100ng/mL(Abcys,Paris,France),humanFMS-liketyrosinekinase3-ligand(hFlt3-L)100ng/mLandhumanmegakaryocytegrowthanddevelopmentfactor(hMGDF)100ng/mL(Amgen,ThousandOaks,CA),mIL-650ng/mL(Abcys),andmIL-1110ng/mL(R&D,Minneapolis,MN).Cellswereseededat1×106/mLinflat-bottomedP96wellscoatedwith50μg/mLrecombinanthumanfibronectinfragment(RetroNectinCH-296;TakaraBiomedicals,Shiga,Japan).Threetransductioncycleswereperformedat24-hourintervalsbyreplacingthemediumwithretroviralsupernatantsupplementedwiththesamecytokinesandprotaminesulfate(4μg/mL)(Choay,Gentilly,France).OnemillioncellswereinjectedintravenouslyintoRAG-1-deficientmicepreviouslyirradiatedat300or800cGyusinga137Csirradiator.Thecontrolgroupwasobtainedbytransplanting1×106wild-typeC57BL/10cellspreviouslyculturedwiththeprotocoldescribedfortransducedcells.MicetreatedwithMFG-RAG-1werekilled4to6monthsaftertransplantation,andsecondarygraftswereperformedasfollows:bonemarrowcellswerecollected,treatedwithanNH4Clsolution,andatotalof5×106cellspermilliliterwereinjectedintravenouslyto300cGy-or800cGy-irradiatedRAG-1-/-recipients. Flowcytometryanalysis Flowcytometryanalyseswereperformedoncellsobtainedfromblood,thymus,bonemarrow,spleen,andlymphnodes.Cellswerecountedwithtrypanblueandstainedwiththefollowingratanti-mousemonoclonalantibodies(MoAbs)(BDBiosciencesPharmingen):fluoresceinisothiocyanate(FITC)-conjugatedCD3(145-2C11),FITC-conjugatedCD25(PC61),PE-conjugatedCD8(53-5.8),PE-conjugatedCD45R/B220(RA3-6B2),allophycocyanin(APC)-conjugatedCD4(L3T4),APC-conjugatedT-cellreceptor(TCRβ)(H57-597),APC-conjugatedCD44(IM7),apolyclonalgoatanti-mouseIgMFITCconjugated(JacksonImmunoResearchLabs,WestGrove,PA),andaratanti-mouseIgDPEconjugated(11-26;SouthernBiotechnologyAssociates,Birmingham,AL).TopreventpossiblebindingtoFcreceptors,peripheralbloodcellswerepreincubatedwithanti-mouseCD16/CD32monoclonalantibodies(MoAbs)(2-4G2;BDBiosciencesPharmingen)andtreated,afterstaining,withfluorescence-activatedcellsorter(FACS)lysingsolution(BDBiosciencesPharmingen)toremoveRBCs.AnalyzeswereperformedonaFACSCalibur(BDBiosciencesPharmingen)usingCellquestsoftware. Lymphocyteproliferationassays Spleenorlymphnodecellswereculturedat1×106/mLinsupplementedDulbeccomodifiedEaglemedium(DMEM;GibcoBRL,Rockville,MD)with10%FBS(GibcoBRL)for3daysinthepresenceofanti-CD3(2-C11;10μg/mL)andanti-CD28(37.51;1μg/mL)antibodies(BDBiosciencesPharmingen)orfor4daysinthepresenceoflipopolysaccharide(LPS;50μg/mL;Sigma-Aldrich).Proliferationwasassayedbyincorporationof[3H]thymidine(2μCi/mL[0.074MBq];Amersham,Saclay,France).Afterovernightincubation,cellsweretransferredontofiltersandplacedin1mLscintillationliquid(PackardBiosciences,Groningen,TheNetherlands).Uptakeof[3H]thymidinewasdeterminedwithascintillationcounter(PackardBiosciences). AnalysisofTCRrepertoire TotalRNAwaspreparedfromsplenocytesusingtheRneasykit(Qiagen,Valencia,CA),andcDNAwassynthesizedusingtheexpandreversetranscriptase(Roche,Indianapolis,IN).ForTCRVβquantitativeimmunoscopeanalysis,cDNAwasamplifiedwitheachof24TCRVβfamily-specificprimerstogetherwithaTCRCβprimerandanMinorGrooveBinder(MGB)-TaqManprobeforTCRCβ.ForTCRVαquantitativeimmunoscopeanalysis,cDNAwasamplifiedwitheachof21TCRVαfamily-specificprimerstogetherwithaTCRCαprimerandanMGB-TaqManprobeforTCRCα.Real-timequantitativepolymerasechainreaction(PCR)wascarriedoutontheABI7300system(AppliedBiosystems,FosterCity,CA).PCRproductswerethensubjectedtorunoffreactionsbyusingnestedfluorescentsprimersspecificfortheCβorCαsegment.Labeledproductswereresolvedonanautomated373Asequencer(PerkinElmer,FosterCity,CA).ThefluorescenceintensityofeachbandwasrecordedandanalyzedusingImmunoscopesoftware(PerkinElmer).24,25  SerumimmunoglobulinquantificationandantibodyresponsetoaT-cell-dependentantigen IgMandIgGimmunoglobulinlevelswereobtainedbyquantitatingserialdilutionsofserumsampleswithanenzyme-linkedimmunosorbentassay(ELISA)kitaccordingtothemanufacturer'sinstructions(BethylLaboratories,Montgomery,TX).T-cell-dependentresponsesagainstkeyholelimpethemocyanin(KLH)weretestedfollowingimmunizationofmiceintraperitoneallywith50μgKLH(Sigma-Aldrich)solutioninalum(Sigma-Aldrich).Micewereboostedwitha50-μgKLHintraperitonealinjection21daysafterimmunization,andserumsamplesweredrawn28daysafterimmunization.Anti-KLH-specificimmunoglobulinsweredetectedbyprecoatingthewellswithKLH(50μg/mL)beforeELISAdetection. Quantificationofgenomeproviralintegrationsandexpression IntegrationstudywasperformedonRAG1-transducedSca-1+cellsandonlymphoidandgranulocytelineagesubsetsisolatedfromspleencells.Briefly,splenocytesfromRAG1-transducedmiceweremarkedwithPE-conjugatedCD4andCD8Abs(145-2C11;BDBiosciencesPharmingen)andAPC-conjugatedGR-1(Ly6-G;BDBiosciencesPharmingen)orbyAPC-conjugatedB220(RA3-6B2;BDBiosciencesPharmingen)andpolyclonalPE-conjugatedIgM(JacksonImmunoResearchLabs).Gr-1+,CD4+/CD8+,andB220+IgM+cellsweresortedwithaFACStar(BDBiosciencesPharmingen)withapurityofatleast99%foreachsubset.GenomicDNAwasextractedbyphenol/chloroformextraction.Real-timequantitativePCRwasperformedwiththeABIPrism7700SequenceDetectorSystem(AppliedBiosystems).AmplificationconditionsconsistedofanAmpliTaqGoldactivationstepat95°Cfor10minutesfollowedby40cyclesof2steps:15secondsofdenaturationat95°Cand60secondsofannealingat60°C.TaqManprobeswereusedfordetectionofthehumanRAG1transgeneandasequencespecificofthemurinegenome(anonrepetitivesequenceofthefifthexonoftheMregionoftitin).Foreachprimerset,astandardcurvewasgeneratedbyserialdilutionofaDNAplasmidcontainingthesequenceamplified.ThedatawerecalculatedusingthestandardcurvemethodgivingPCRefficiencyforeachprimersetandattributingvaluesforeachsamplerelativetothereferencesamplecontrol.Allsamplesandserialdilutionswereruninduplicate.Thereferencecontrolwasaplasmidcontainingbothsequences(titinandRAG1),andtheresultsobtainedfromtitinquantificationwereusedtonormalizethenumberofcopiesbacktothenumberofcells. Theprimersandprobesequencesusedareasfollows:1894MFGRAG1.forward,GGTGGACCATCCTCTAGACTGC;1979MFGRAG1.reverse,TGGGTGCTGAATTTCATCTGG;1920MFGRAG1.probe,CAGCCTCTTTCCCACCCACCTTGG;m139Mex5.forward,AAAACGAGCAGTGACGTGAGC;m261Mex5.reverse,TTCAGTCATGCTGCTAGCGC;M161TitinMex5.probe,TGCACGGAAGCGTCTCGTCTCAGTC. hRAG1transgeneexpressionwasquantifiedinthesamplesobtainedfromvarioustissueslikespleen,thymus,bonemarrow,orlymphnodes.RNAwasextractedwiththeRneasyminikit(Qiagen,Hilden,Germany).OnemicrogramoftotalRNAwasreversetranscribedtosingle-strandedcDNAwith50unitsofSuperscriptIIReagent(Invitrogen,Carlsad,CA)andrandomhexamers.Reversetranscription(RT)wasperformedina20-μLfinalvolume,andthesamplewasincubatedat42°Cfor60minutes.ToaccountforvariationsduetoRNAextractionandtheRTreaction,themeasuredlevelofhRAG1transgenemRNAwasnormalizedagainstanendogenousRNAcontrol,POmRNA,anacidicribosomalphosphoproteinubiquitouslyexpressedinallmurinecells.26 RatioswerecalculatedbycomparingthehRAG-1mRNAexpressionlevels(hRAG-1/PO)inthetestedsampleswithmurineRAG-1expressionlevelsinthethymusofwild-typeC57BL/10mice(mRAG-1/PO).ThehRAG-1transcriptexpressionvaluewascalculatedasapercentageofthereferencemRAG-1/POratiointhethymus:%hRAG-1expression=(hRAG-1/PO)/(mRAG-1/PO)×100. Theprimersandprobesequencesusedareasfollows:678hRAG1.forward,TCAGCCAAACTTGCAGCTCA;780hRAG1.reverse,CTTGCTGCTGATCCTTGCCT;722hRAG1.probe,ACCAAGCAAGACAAGCCCGTCAGC;MH181PO.forward,CTCCAAGCAGATGCAGCAGA;M267PO.reverse,ATAGCCTTGCGCATCATGGT;M225PO.probe,CCGTGGTGCTGATGGGCAAGAA. Figure1.ViewlargeDownloadPPTThymusprofileafterRAG1retroviral-mediatedgenetransferintoRAG-1-deficientmice.Micereceivingtransplantswerekilled6monthsafterRAG1genetransfer.FlowcytometryanalysiswasperformedontotalthymiccellsforCD4andCD8staining.FortheCD25andCD44distributionanalysis,thedot-plotwindowwasgatedontheCD4-CD8-cells.Thedotplotsarerepresentativeofoneexperiment:n=4fortheRAG-1-deficientmice(RAG-1-/-),n=5fortheRAG1-transducedmice(RAG1-TM,3Gy),n=4fortheRAG1-transducedmice(RAG1-TM,8Gy),n=8forthecontrolmice(WT-CTRL),andn=4forthesameagewild-typeC57BL/10mice.PercentagesrepresenttheproportionofcellsexpressingCD4,CD8,CD25,orCD44markers.Figure1.ViewlargeDownloadPPTThymusprofileafterRAG1retroviral-mediatedgenetransferintoRAG-1-deficientmice.Micereceivingtransplantswerekilled6monthsafterRAG1genetransfer.FlowcytometryanalysiswasperformedontotalthymiccellsforCD4andCD8staining.FortheCD25andCD44distributionanalysis,thedot-plotwindowwasgatedontheCD4-CD8-cells.Thedotplotsarerepresentativeofoneexperiment:n=4fortheRAG-1-deficientmice(RAG-1-/-),n=5fortheRAG1-transducedmice(RAG1-TM,3Gy),n=4fortheRAG1-transducedmice(RAG1-TM,8Gy),n=8forthecontrolmice(WT-CTRL),andn=4forthesameagewild-typeC57BL/10mice.PercentagesrepresenttheproportionofcellsexpressingCD4,CD8,CD25,orCD44markers. LAM-PCRanalysis Anintegrationsiterestrictionfragmentlengthdisplaywasobtainedbylinearamplification-mediatedPCR(LAM-PCR)consistingofrepeatedprimerextension,secondstrandsynthesis,restrictiondigest,cassetteligation,andexponentialamplificationasdescribedpreviously.27 LAM-PCRwasperformedonDNAisolatedfromspleen,bonemarrow,orlivercells,andthefinalproductwasseparatedonSpreadexgels(Elchrom,Cham,Switzerland)tovisualizethepolymorphismofintegrationssites. Statisticalanalysis Mann-WhitneyUtestswereusedtocompareimmunereconstitutionbetweenRAG1-transducedmiceandcontrolmice.DatawereconsideredtobestatisticallysignificantatPvalueslessthan.05. Results ImmunereconstitutionfollowingexvivoRAG1genetransfer Sixmonthsfollowinggenetransfer,T-celldevelopmentwasanalyzedintheRAG1-transducedmice(RAG1-TM,n=9)aswellasinRAG-1-/-controlmicethatreceivedcongenicwild-typebonemarrowcells(WT-CTRL,n=8).Intherecipientmiceirradiatedwitha8Gydose(n=4),thymocytecountsandthymicreconstitutionfeatureareundistinguishablefromWT-CTRLmice(Figure1;Table1).Inthe3Gy-irradiatedgroup(n=5),overallthymocytecountsweremorevariable(from1×105cellsto8×106cells)andlowerthanthethymocytecountsobservedinthe8Gy-irradiatedgroup(P<5%).Ofnote,CD4+CD8+double-positive(DP)cellswerefoundinonly2of5recipientsalthoughmaturesingle-positive(SP)thymocytes(CD4+CD3+orCD8+CD3+)weredetectedinallcases.InbothRAG1-TMandWT-CTRLgroups,thepresenceofDPcellswasassociatedwithanormaldistributionofthedouble-negativethymocytesubsetsasstudiedbytheCD25andCD44surfaceantigenexpression(Figure1).Together,thesedatademonstratethatthymusreconstitutionfollowingexvivoretroviralgenetransferisdirectlydependentontherecipients'irradiationdose.Thespleen,lymphnode,andbonemarrowcellcountsweresimilarinallgroups—thatis,RAG1-TM(3or8Gyirradiated),WT-CTRL,andwild-typeC57BL/10mice(Table1).However,theproportionofmatureBlymphocytes(B220+IgM+IgD+)wasreducedinthelymphoidorgansofRAG1-TMmicewhencomparedwithbothcontrolgroups.SuchadifferencewasnotobservedinthematureT-cellcompartment(Figure2).InalltheRAG1-TMmice,circulatingmatureTcellsbecamedetectable6weeksaftergenetherapywithaslightsteadyincreaseduringthefirst4monthsofreconstitution.T-cellcountsandtheCD4/CD8ratioreachedWT-CTRLlevelswithin6monthsandremainedstableupto1yearaftergenetherapy(Figure3A,C).Thesecellcountswereverysimilartothosedetectedinage-matchedwild-typeC57BL/10mice,attestingthattheT-cellperipheralpoolwasfullyreconstituted.Incontrast,bloodB-lymphocytereconstitutionwasdelayedinRAG1-TMmice,andB-cellcountsremainedpersistentlylowerthanincontrolmice(Figure3B-C).Ofnote,B-cellcountsinWT-CTRLmicewerelowerthaninage-matchedwild-typeC57BL/10mice(1883±372cells/mm3and4300±809cells/mm3,respectively)even1yearaftergenetherapy.Unlikethedifferencesobservedbetweenthymicreconstitutioninthe3Gy-and8Gy-recipientmice,cellcountsandlymphocytesubsetdistributionweresimilarinthelymphoidorgansandintheperipheralbloodofbothirradiatedrecipientgroups. Table1.LymphocytecountsintheorgansofRAG-1-deficientmice,RAG1-transducedmice,controlmice,andwild-typeC57BL/10mice6monthsafterinjectionofexvivo-transducedSca-1cells . Thymus,×106cells . Lymphnodes,×106cells . Spleen,×106cells . Bonemarrow,×106cells . RAG-1–/–;n=4 0.2±0.1 3±2 4±1 35±11 RAG1-TM,3Gy;n=5 3±4 15±3 35±8 36±11 RAG1-TM,8Gy;n=4 11±4 14±5 33±14 34±3 WT-CTRL;n=8 14±5 18±6 28±6 30±5 C56BL/10;n=4 75±27 15±6 36±13 45±12  . Thymus,×106cells . Lymphnodes,×106cells . Spleen,×106cells . Bonemarrow,×106cells . RAG-1–/–;n=4 0.2±0.1 3±2 4±1 35±11 RAG1-TM,3Gy;n=5 3±4 15±3 35±8 36±11 RAG1-TM,8Gy;n=4 11±4 14±5 33±14 34±3 WT-CTRL;n=8 14±5 18±6 28±6 30±5 C56BL/10;n=4 75±27 15±6 36±13 45±12 Dataareexpressedasmean±SD.TheMann-WhitneyUtesthasbeenusedtocomparethelymphocytecountsineachgroup.Aftergenetherapy,thecomparisonbetweenthethymocytecountsinthe3Gy-and8Gy-irradiatedrecipientisstatisticallysignificant(P<.05 figure2.viewlargedownloadpptt-andb-lymphocytereconstitution.reconstitutionisdepictedinlymphnodes t-andb-cellcharacteristicsaftergenetherapy toanalyzethev figure3.viewlargedownloadpptbloodlymphocytecountsafterrag1retroviral-mediatedgenetransferinrag-1-def icientmice.peripheraltlymphocytes figure4.viewlargedownloadpptb-andt-cellcharacteristicsafterrag1retroviral-mediatedgenetransferintora g-1-deficientmice. provirusintegrationandexpressionofthehrag1transgene toquantifytheintegrationandexpressionofthehumanrag1transgene stabilityoftransgeneexpression rag1-tmmicewereanalyzedformorethan1yearaftertreatment.atthelasttimepoint figure5.viewlargedownloadpptquantificationofprovirusintegrationandhrag1transgeneexpression. toxicity sixmonthsaftergenetherapy figure6.viewlargedownloadpptanalysisofsecondarytransplantationinrag-1- discussion rag-1-immunodeficientmicewereusedasamodeltodemonstratethatexvivorag1retroviralgenetransferintorag-1- figure7.viewlargedownloadpptcharacteristicsofthesevereadverseeffectobservedinonemouseafterrag1genetr ansfer.sixmonthsafterprimarytransplantation itisnoteworthytostressthatimmunologicreconstitutionwasonlyobservedwhenahighcopynumberofhrag1transgen epercellcouldbedetected.ahighcopynumbermayberequiredtocompensateforalowtranscriptionrateofhrag1trans gene aselectiveadvantageofrag1-transducedcellsovernontransducedcellswasclearlyobserved thehighcopynumberrequiredtorestoretherag-1immunodeficiencyledtotheoccurrenceofonecaseofclonallymphop roliferationamongthe30genetherapy-treatedmice.thelymphoproliferationhadsomecharacteristicsofanacutel ymphoblasticleukemia.itwasstrikingthatrag-1expressionwasnotenhancedbutmrag-2expressionwasdetectedint hetumorcells.thisislikelytheconsequenceoftheimmaturelymphocyteprogenitorstageoftheproliferativecells .onecannot onepotentialconcernofrag1genetherapyisthepotentialtoxicityofuncontrolledrag-1expressiondrivenbyacont inuouslyactivepromoter.physiologically inconclusion prepublishedonlineasbloodfirsteditionpaper supportedbygrantsfrominserm theonlineversionofthisarticlecontainsadatasupplement. thepublicationcostsofthisarticleweredefrayedinpartbypagechargepayment.therefore theauthorsthankfabiangrossfortechnicalhelp copyright supplementaldata figures1 figures2.murinerag-2 addcomment closecommentformmodal submitacomment name affiliations commenttitle comment youmustacceptthetermsandconditions. youhaveenteredaninvalidcode submit cancel thankyouforsubmittingacommentonthisarticle.yourcommentwillbereviewedandpublishedatthejournal close thisfeatureisavailabletosubscribersonly signin or createanaccount closemodal volume107 january12006 previousarticle nextarticle advertisement citations viewmetrics citedby webofscience googlescholar emailalerts articleactivityalert latestissuealert currentissue firstedition allissues collections abstracts authors submittoblood aboutblood subscriptions publicaccess permissions alerts contactus bloodclassifieds advertisinginblood termsandconditions twitter americansocietyofhematology washington tel fax ashpublications blood bloodadvances hematology ashclinicalnews ash-sap thehematologist ashhome research education advocacy meetings publications ashstore privacypolicy cookiepolicy termsofuse signinorcreateanaccount x>