Asurveyof exvivoinvitro transductionefficiency ofmammalianprimarycellsandcelllineswith NinenaturaladenoassociatedvirusAAV19and oneengineeredadenoassociatedvirusserotype BrianLEllis 1 MatthewLHi ID: 447295
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RESEARCHOpenAccess Asurveyof exvivo/invitro transductionefficiency ofmammalianprimarycellsandcelllineswith Ninenaturaladeno-associatedvirus(AAV1-9)and oneengineeredadeno-associatedvirusserotype BrianLEllis 1 ,MatthewLHirsch 2,3 ,JennyCBarker 1 ,JonPConnelly 1 ,RobertJSteiningerIII 4 andMatthewHPorteus 1,5* Abstract Theabilitytodeliverageneofinterestintoaspecificcelltypeisanessentialaspectofbiomedical research.Virusescanbeausefultoolforthisdelivery,particularlyindifficulttotransfectcelltypes.Adeno- associatedvirus(AAV)isausefulgenetransfervectorbecauseofitsabilitytomediateefficientgenetransductionin numerousdividingandquiescentcelltypes,withoutinducinganyknownpathogenicity.Therearenowanumber ofnaturalforthatdesignedAAVserotypesthateachhasadifferentialabilitytoinfectavarietyofcelltypes. Althoughtransductionstudieshavebeencompleted,thebulkofthestudieshavebeendone invivo ,andtherehas neverbeenacomprehensivestudyoftransduction exvivo/invitro . Methods: Eachcelltypewasinfectedwitheachserotypeatamultiplicityofinfectionof100,000viralgenomes/cell andtransductionwasanalyzedbyflowcytometry+. Results: WefoundthatAAV1andAAV6havethegreatestabilitytotransduceawiderangeofcelltypes,however, forparticularcelltypes,therearespecificserotypesthatprovideoptimaltransduction. Conclusions: Inthiswork,wedescribethetransductionefficiencyoftendifferentAAVserotypesinthirty-four differentmammaliancelllinesandprimarycelltypes.Althoughtheseresultsmaynotbeuniversaldueto numerousfactorssuchas,cultureconditionsand/orcellgrowthratesandcellheterogeneity,theseresultsprovide animportantanduniqueresourceforinvestigatorswhouseAAVasan exvivo genedeliveryvectororwhowork withcellsthataredifficulttotransfect. AAV,Serotypes,Adeno-associatedvirus,Genetherapy,Tropism,Primarycells,Progenitorcells,Celllines, Transduction, exvivo Background Afundamentaltechniqueinbiomedicalresearchisto deliverageneofinterest(transgene)intoacellinorder toalteritsbehavior.Whiletransgenedeliverycanbe achievedbyanumberofdifferenttransfectionstrategies, suchaschemical,lipid,orelectroporationbasedmethods, therearemanycelltypesthatarenotefficiently transfectedintheseways(forexampleprimaryT-cells, cardiomyocytes,andprimaryhematopoieticstemcells). Viralvectorshavebecomeanimportantresourcetoover- comethesebarrierstogenetransfer.Thereareanumber ofdifferentviralvectorsthathavebeenusedforgene transfer,includingretroviruses,lentiviruses,andadeno- virus,butoneofthemostutilizedviralvectorshasbeen recombinantadeno-associatedvirus(AAV).WhileAAV serotype2(AAV2)hasbeenthemostwidelyused AAVcapsidvariants,eachofwhichhasadifferenttropism *Correspondence: mporteus@stanford.edu 1 DepartmentofBiochemistry,UniversityofTexasSouthwesternMedical Center,Dallas,TX,USA 5 DepartmentofPediatrics,UniversityofTexasSouthwesternMedicalCenter, Dallas,TX75390-9148,USA Fulllistofauthorinformationisavailableattheendofthearticle ©2013Ellisetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited. Ellis etal.VirologyJournal 2013, 10 :74 http://www.virologyj.com/content/10/1/74 fordifferentcelltypes[1,2].Thus,AAVisagenerallyuse- fulvectorforgenetransferinawiderangeofcelltypes. AAVisasmall,non-envelopedvirusthatpackages bothnegativeandpositivepolaritysingle-strandedDNA. AAVisamemberoftheParvoviridaefamilyandre- quiresahelpervirus,suchasadenovirusorherpesvirus, foraproductiveinfection.Thewild-typegenomeis 4.7kbandcontainstwomajoropenreadingframes (ORFs)thatincludetheRepgeneandCapgene.In addition,athirdORFwasrecentlyshowntoexist[3]. WhenAAVisusedasagenetransfervector,the endogenousgenesareremovedandreplacedbyan expressioncassetteforthegeneofinterest.Oneofthe barrierstoefficientexpressionofthetransgeneisthe conversionofthesingle-strandAAV(ssAAV)genome intoaduplexedsingleDNAmolecule[4].Thelimitation intransgeneexpressionfromssAAVvectorshasbeen improvedbythedevelopmentofself-complementary AAV(scAAV)vectorsinwhichthesingle-strandedAAV genomeself-hybridizestoformduplexDNA(Figure1). scAAVvectorshaveshownearlieronsetoftransgeneex- pressionandoverallhighertransductionefficienciesthan ssAAVvectors[4,5]. Theabilitytotransducedifferentcelltypesisprimarily determinedbytheAAVproteincapsid[1].Thedifferent capsidsbindtodifferentcellularreceptorsandthisbind- ingmediatesentryintothecell.Theprimaryreceptor forAAV2andAAV3isheparansulfate-proteoglycan[6]. Integrin 5 5,integrin 5 1,hepatocytegrowthfactor receptor(c-Met),andCD9havealsobeendescribedpre- viouslyaspotentialco-receptorsforAAV2[7-10].The fibroblastgrowthfactorreceptor-1isaco-receptorfor bothAAV2andAAV3[11]andthe37/67-kDalaminin receptorisaco-receptorforAAV2,AAV3,AAV8and AAV9[12,13].TheprimaryreceptorforAAV1,AAV4, andAAV5isO-linkedsialicacid,whiletheprimaryre- ceptorforAAV6isN-linkedsialicacid[14-17].The plateletderivedgrowthfactorreceptorisaco-receptor forAAV5[18].Theconsequenceofthedifferentcellular receptorsforcapsidbindingisthateachofthesenatural AAVserotypestransducesadifferentrangeofcelltypes. TheabilityofdifferentAAVserotypestotransducedif- ferentcelltypeshasbeenpreviouslyreported,butmost ofthesestudieshavebeendone invivo ,andtheyreport ontheeffectivenessoftissuetypetransductionrather thancelltypetransduction[2].Forexample,abroad studyofAAVserotypes1-9hasbeendoneinmice[19]. However,acomplete exvivo/invitro studyoftransduc- tionefficiencyislacking.Inthiswork,weperformedan extensivesurveywherethirty-fourdifferentmammalian celltypesweretransducedwithtendifferentAAVsero- types exvivo/invitro .Thiscollectivedataprovidesan importantanduniqueresourcetotheresearchersinter- estedingenedelivery exvivo andinculturedcelllines. Ourdataclearlydemonstratethatthereareclearquali- tativedifferencesfortheabilityofdifferentserotypesto transducedifferentsub-typesgivinggeneralguidanceon thebestserotypestouseandthat apriori predictionis notalwayspossible.Transductionvariabilitycouldbe high,particularlywhentheinfectionefficiencyislow, andsuggestthatthedatashouldbegenerallyviewedin 7broadcategories:1:0%,2:�0-1%,3:1-10%,4:10-30%, 5:30-60%,6:60-80%,7:80-100%.Moreover,thesecat- egoriesshouldnotbeviewedasrigidasitislikelythat transductionof8%,forexample,isnotnecessarily differentthan12%. Resultsanddiscussion ToanalyzethetropismofninedifferentnaturalAAVse- rotypes(1-9)andoneengineeredserotype(1.3)(ahybrid ofAAV1andAAV6),weusedscAAVvectorsthat expressedeGFPfromtheCMVpromoter(Figure1). EventhoughssAAVhasalargercloningcapacitythan scAAV,wechosescAAVbecauseoftheoverallimproved transgeneexpressionofitsvectorscomparedtossAAV vectorsasthisreportwasintendedtobeastraightfor- wardcapsidcomparison.Becausesomecellshavebeen reportedtoberefractorytoAAVtransduction,we wantedtousethemostefficientgenometechnology helpingtoreducethepossibilitythattimingandamount oftransgeneexpressionwouldbiastheresults.We selectedeGFPasatransgenebecauseoftheeaseof quantitatingtransgeneproductfluorescencebyflow cytometryandbecauseliveculturescouldbeanalyzed bymicroscopy.Weinfectedallcelllinesataconstant multiplicityofinfection[MOI(definedhereasvector genomespercell)]of100,000vectorgenomes/celland analyzedforeGFPexpressiontwodaysafterinfection. Furthermore,werepeatedtheinfectionsatanMOIof 10,000andsawthesametrends,thoughalowerper- centageofGFP+cells(datanotshown).Although, CMV poly A eGFP Figure1 Aschematicrepresentationoftheself-complimentary AAV(scAAV)genome. WeconstructedascAAVgenome(double blacklines)thatincludedaneGFPreportergene(ingreen)drivenby theCMVpromoter(blackarrow)thatallowedforefficientand quantitativeanalysisoftransduction.Thisconstructalsoincludeda polyAsequence(blackline)andinvertedterminalrepeats(ITRs) inblue)). Ellis etal.VirologyJournal 2013, 10 :74 Page2of10 http://www.virologyj.com/content/10/1/74 MOIsof10,000or100,000insomecasesmightbe consideredhigh,MOIsof10,000andupto500,000have beenusedforgenetargeting[20,21],andimportantly,it ensuresthatifacellwasnottransduceditwasnot becausetoolowofanMOIwastested.Theresultsare presentedasheat-maps;highertransductionefficiencies (measuredas%GFP+cells)aredisplayedinred,and lowertransductionefficienciesareinblue.Theactual transductionefficiencyisgivenasapercentage.A completelistofthecellstransducedinbothFigures2 and3arepresentedinTable1andadescriptionofthe isolationoftheprimarycellsarelistedintheMaterials andMethodssection. Transductionofhumanprimarycells WeevaluatedtendifferentAAVserotypesfortheir abilitytotransducesixdifferentpurifiedprimaryhuman celltypes:BJfibroblasts,BJhTERTfibroblasts,em- bryonicstemcells(ES),humanumbilicalcordvein endothelialcells(HUVEC),humankeratinocytes,and humanhematopoieticprogenitorcells(Figure2a).To avoidheterologousmixturesofcells,theprimarycell typeswereeitherisolated,asdescribedpreviously(see MaterialsandMethods),orpurchasedaspurifiedcells. At48hourspostinfection,wefoundbasedon%GFP+ cells,thatAAV1,2and6besttransducedhumanfibro- blasts,AAV3wasmostefficientforhumanEScells, AAV1,1.3,2,and6showedthehighesttransductionfor HUVECs,andAAV1,1.3and6besttransduced keratinocytes.Wefoundnoneoftheseserotypeseffi- cientlytransducedhumanhematopoieticprogenitorcells (purifiedCD34+cells).WenotetheBJfibroblasts,BJ hTERTfibroblasts,EScells,andHUVECcellsarenot freshlyisolatedcells.However,wecategorizethemas primarycellsherebecausetheyarenottransformedand showthesamepropertiesasfreshlyisolatedcells. Transductionofhumancelllines InFigure2b,wereportourresultsforthetransduction oftwelvedifferenthumanderivedcelllines48hours postinfectionbasedon%GFP+.WefoundthatCaco-2 (anepithelialcolorectalcelltype)andK562cells (ahematopoieticderivedcellline)werenotefficiently transducedbyanyoftheAAVserotypes,although20% A B Figure2 scAAVtransductionofhumanprimaryandimmortalizedcells.A) Humanprimarycellsand B) humanimmortalizedcelllineswere transducedwitheGFPscAAVatamultiplicityofinfection(MOI)of100,000viralgenomes(vg)/cell.Thecellswereanalyzedbyflowcytometryat 48hourspost-infectionforthepercentagethatwereGFPpositive.ThenumberintheboxistheactualpercentageofGFPpositivecellswiththat serotype.*=Transductionlessthan0.01%butgreaterthan0.0%. Ellis etal.VirologyJournal 2013, 10 :74 Page3of10 http://www.virologyj.com/content/10/1/74 ofTF1- cells(adifferenthematopoieticcellline)were GFP+usingAAV2.ThelackoftransductionofK562 cellscannotbeexplainedbythelackofexpressionfrom aCMVpromoter,asstrongCMVmediatedtransgene expressionisobtainedaftertransfectionofK562cells (datanotshown).Alloftheremainingcelltypeswere transducedtoatleast11%,andmostweretransducedat muchhigherefficiencies.Overall,AAV1andAAV6were twoofthebestserotypesforefficienttransductionof humancelllines,whileAAV2andAAV3werealso broadlyeffectiveintransductionofhumancelllines (Figure2b). Transductionofmurineprimarycells Wetransducedninedifferentprimarymurinecelltypes (adultskinfibroblasts,astrocytes,EScells,hematopoietic progenitors,keratinocytes,mesenchymalstemcells, murineembryonicfibroblasts(MEF),skeletalmuscle progenitor,andwhiteadiposeprogenitorcells)withthe tendifferentAAVserotypes.Wealsotransducedtwo differentprimarymurinecelltypes(murinelungepithe- lialandlungmesenchymalcells)withAAV6.Wethen evaluated%GFP+at48hourspostinfection(Figure3a). Allofthesemurineprimarycellswereeitherisolated,as describedpreviously(seeMaterialsandMethods),or purchasedaspurifiedcells,avoidingheterologousmix- turesofcells.Wefoundthatnoneoftheserotypeseffi- cientlytransducedmesenchymalstemcellsorskeletal muscleprogenitors.WeshowedthatAAV6wasthebest serotypefortransducinghematopoieticprogenitorcells, butonlytoalevelof10%,arelativelylowpercentage (Figure3a,row4).Incontrasttomostothercelltypes tested,wefoundthatAAV4infectswhiteadiposepro- genitorcellsexceptionallywell,especiallyincomparison toallotherserotypes(Figure3a,row11).Wesawthat murineEScellstransducewellwithAAV1(Figure3a, row3,25%).ThisalsofitswiththedatatheMcWhir groupshowedusingAAV2,4,and5thatmEScellswere nottransducedefficiently[22].Fortheremainingcell types,atleastoneAAVserotypewasefficientinmediat- ingtransduction.Ingeneral,AAV1andAAV6weretwo ofthebestserotypesfortransductionofmouseprimary cellsundertheconditionstested. Transductionofmonkey,hamster,andmousecelllines Wetransducedfivedifferentmammaliancelllines,in- cludingmurine3T3cells,murineC2C12cells,murine MIN6cells,Chinesehamsterovarycells(CHO),and monkeyCOS-7cellswiththetendifferentAAVsero- typesandevaluated%GFP+at48hourspostinfection A B Figure3 scAAVtransductionofmurineprimarycellsandmurine,hamster,andmonkeyimmortalizedcells.A) Murineprimarycellsand B) murine,hamster,andmonkeyimmortalizedcelllinesweretransducedwitheGFPscAAVatamultiplicityofinfection(MOI)of100,000vg/cell. Thecellswereanalyzedbyflowcytometryat48hourspost-infectionforthepercentagethatwereGFPpositive.Thenumberintheboxisthe actualpercentageofGFPpositivecellswiththatserotype. Ellis etal.VirologyJournal 2013, 10 :74 Page4of10 http://www.virologyj.com/content/10/1/74 (Figure3b).Everycelllinewetestedhadatleastoneserotypethattransducedcellswith�50%transductionefficiency(Figure3b).Insomecasesourresultsdidnotmatchperfectlywithpreviousresults[23](forexampleAAV1,AAV2,AAV5onC2C12cells;AAV1,AAV2,AAV5onCHOK1cells).WiththeexceptionofAAV5onC2C12cells,thedatapresentedherereasonablyreflectpreviousdata[23]whenviewingthepanelquali-tatively,andthedifferencesincultureconditionsandMOI(10,000or100,000vs.300vg/cell)couldaccountforthevariability.Anotherdiscrepancyfromourdata[24]showedthatAAV2transducedCOScellsbetter Table1CelltypesanddescriptionHumanPrimaryCellsBJFibroblastsForeskinfibroblastsBJhTERTFibroblastsForeskinfibroblastsretrovirallyinfectedwithhTERTEScellEmbryonicstemcellsHumanumbilicalcordveinendothelialcellsKaratinocytesKeratinocytesHematopoieticProgenitorCD34+umbilicalcordcellsHumanCellLinesEpithelialcolorectaladenocarcinomacellsHumanbronchialepithelialcellsHEK293HumanembryonickidneycellsCervicalcancercellsHepatocellularcarcinomaColonadenocarcinomagradeIIcellsimmortalizedlineofTlymphocytecellsMyelogenousleukemiacellsBreastcancercellsErthroleukemiccellsOsteosarcomacellsOsteosarcomacellsMousePrimaryCellsAdultSkinFibroblastMurineadultskinfibroblasts(MAFs)EScellEmbryonicstemcellsHematopoieticProgenitorcKit+,Sca+,Lin-hematopoieticcellsKeratinocytesKeratinocytesLungEpithelialEpithelialcellsLungMesenchymalMesenchymalcellsMesenchymalStemCellsMesenchymalstemcellsEmbryonicFibroblastMurineembryonicfibroblasts(MEFs)SkeletalMuscleProgenitorSkeletalmuscleprogenitorcellsWhiteAdiposeProgenitorWhiteadiposeprogenitorcellsMouseCellLines3T3HeterogeneousembryonicmousecellsMyoblastcellsPancreaticbetacellsOtherCellLinesChinesehamsterovarycellsAfricangreenmonkeykidneyfibroblastsListedarethecelltypestransducedbyAAV1-9andAAV1.3inFigures.TheMaterialsandMethodssectionlistamoredetaileddescriptionoftheprimarycellisolations.etal.VirologyJournalPage5of10http://www.virologyj.com/content/10/1/74 thanAAV1.Althoughthedifferenceisdramatic,itcouldpossiblybeexplainedbychangesinviralpreparation,cultureconditions,andthedetectionsystemused(hFIXexpressionvs.GFPexpression).Overall,wefoundthatAAV1andAAV6aretwoofthebestserotypestoinfectcelllinesofmouse,hamster,andmonkeyoriginundertheconditionstested;however,theparticularcelltypemustbeconsidered,asotherserotypesweremoreefficientinsomecases.ExpansioninthenumberofAAVserotypes,boththroughtheidentificationofnovelnaturalserotypesandnew,engineeredserotypes[16,23,25-35],hasresultedinimprovedgenetransfertospecificcelltypesinvivoTherehavebeenseveralpublicationsreviewingthepreferredinvivotropismofthesenewserotypes[2,36].Howeverexvivo/invitrodataislacking.AlternativeusesofAAVincludeusingthisviralvectorexvivoasamethodofgenetransferintospecificcelltypes.Thesetransducedcellscouldthenbestudieddirectlyorusedforcell-basedgenetherapy,wherebyAAVtransductionwouldoccurandthemodifiedcellswouldthenbetransplanted.AnotherpotentialuseofAAVvectortransductionexvivowouldbetouseAAVvectorstostimulategenetargetingbyhomologousrecombination,topreciselymodifythegenomeofthecellstobetransplanted.ThismodificationcouldbedonethroughgenetargetingdirectlybyAAV[37-40]orincombinationwiththeinductionofasite-specificdouble-strandbreak.Thesesite-specificdouble-strandbreakscouldbeinducedbyahomingendonuclease[21,41,42],byzincfingernucle-ases[43],orbysomeothernuclease,likeTALeffectornucleases(TALENs)[44-47].AnimportantaspecttousingAAVinthismanneristodeterminethebestserotypetotransducespecificcelltypesexvivo,wherethereisnobasementmembraneorextracellularmatrix.Infact,wehavealreadyreportedtheabilityofasingleAAV6vectortodeliverbothzinc-fingernucleasesaswellasadonorrepairsubstrate,tostimulategenetargeting[20].Inregardstostemcells,itisintriguingthatmanypro-genitorcellsdidnottransducewell(i.e.seelotsofdarkblueintheheatmaps).Perhapsthecellshaveevolvedtheabilitytoavoidtransductionasawaytoprotectthemselvesfromchangestothecell,inparticulartheDNA.However,therecertainlywerestemcellsthatweretransducedwellbyvariousAAVserotypes.Thisagainpointstotheutilityofthisstudy,ascertainserotypesweregoodfortransductioninsomestemcellsandbadinothersandalthoughAAV1andAAV6weregoodorthebestattransductioninmanystemcells,thereweresomestemcellsthattransducedpoorlywithAAV1andAAV6.InthisworkweprovideabroadsurveythatexaminestheabilityoftendifferentAAVserotypestoinfectthirty-fourdifferentcelltypesexvivo/invitro.Ingeneral,wedemonstratethatAAV1andAAV6havethegreatestabilitytotransduceawiderangeofcelltypes.Wefound,however,thatforparticularcelltypestherearespecificserotypes,whichprovideoptimaltransduction.(Forex-ample,AAV4istheoptimalserotypefortransducingmurineadiposeprogenitorcells.)Wealsofoundthattherearecertainprimarycelltypes,suchashumanhematopoieticprogenitorcells,thatwerenotefficientlytransducedbyanyofthetendifferentserotypes.ItispossiblethatthelackofmeasuredtransductioninthesecelltypesisbecausetheCMVpromoterisrelativelyweakinthesecells.Itisnotlikely,however,thatinthesecasesthecellswereoverloadedwithuptakeandprocess-ingofvirionsbecausewhena10-foldlowerMOIwasused,lowertransductionefficiencywasseenineverycase(datanotshown).Although,differentgrowthcondi-tionswereusedformanyofthedifferentcelltypes,eachserotypewasusedforeachcelltypeinthesamecondi-tions,thusprovidinganinternalcontrolforacompara-tiveanalysis.However,becauseasmallamountofourdatadoesnotperfectlymatchwithpreviousfindings,wesuggestthattheresultspresentedhereshouldleadin-vestigatorstochooseafewofthebestserotypesfortheirspecificneed.Ourresultsdemonstratethatthereisnosimplemechanismtopredictwhichserotypewilltrans-duceaparticularcelltypebutdoessuggestthatifonewerelimitedtoscreeningasmallnumberofserotypesthatfocusingonAAV6,AAV2,andAAV3wouldberea-sonableasthosethreeserotypesgiveabroadrangeofef-fectivenessacrossmostcelltypes.Inthefuture,itmaybeimportanttofurtherstudythetransductionofAAVafterdifferentpurificationstrategiesareused,asithasbeenshowntoaffecttransduction[48].Methodstofa-cilitateAAVtransduction,suchasbytheuseofprote-asomeinhibitors[49]orstrategiesthatallowforselectionofnovelcapsids,mayhelpovercomethebar-riertotransductionthatthesecellsexhibit.However,itislikely,therearefactors,unprovenasofyet,thatserveasmajorbarrierstotransductionbyAAV.Forexample,itispossiblethattheapparentlowtransductioncouldbeaconsequenceofAAVvectorsinducingapopto-sis[50].Inthiscase,acaspaseinhibitorsuchasZ-VAD-FMKcouldbeusedtoachievetransductionwithoutcelldeath.Understandingtheseunprovenbar-rierstotransductionwouldfurtherimprovetheutilityofAAVasagenetransfervectorforexvivomanipulationofprimarycellsaswellasinvivogenetherapy.Insummary,wehaveperformedasurveyoftheabilityofdifferentAAVserotypestotransduceawidevarietyofetal.VirologyJournalPage6of10http://www.virologyj.com/content/10/1/74 differentprimaryandimmortalizedcelltypes.ThissurveyshouldbeausefulandpracticalresourceforinvestigatorsastheyconsiderusingAAVasagenetrans-fervectorintheirstudies.EthicsstatementAllanimalworkhasbeenconductedaccordingtorele-vantnationalandinternationalguidelines.ApprovalforstudiesusingcellsderivedfrommicewasobtainedfromtheUTSouthwesternIACUC,APB#2010-0106.AAVproductionWethankR.J.Samulskiforprovidingtheself-complementaryeGFPandthepXRseriesofplasmidsusedherein.AAVvectorproductionreliedonthetripletransfectionmethoddescribedpreviously[51].Briefly,cellsweretransfectedwiththeadenovirushelperplasmidpXX680,pHpa-Trs-SKCMV-eGFP(togenerateself-complementaryAAVgenomes)[5],aplasmidthatcodesforAAVRep2,andaspecificcapsidserotype(pXRseries1-9,correspondingtoAAVserotypes1-9).ThreedaysafterHEK293celltransfectioninaplateformat,nucleiwereharvested,disrupted,andthelysatewasseparatedbycesiumchloridegradientcentrifugation[51].DNasewasusedduringpurification.Followinganovernightspinat55,000rpm,12gradientfractionswerepulled.Todeter-minethegradientfractioncomposedofpurescAAVge-nomes,10LofeachfractionwassubjectedtoSouthernblottingfollowingalkalinegelelectrophoresisaspreviouslydescribed[51].FractionscontainingonlyscAAVgenomeswerepooled,dialyzedagainst1XPBS,aliquotedandstoredat-80°Cuntiluse.FinaltiterdeterminationwasperformedaftertheinitialthawbyquantitativePCRusingprimersspecificfortheeGFPtransgene(forwardprimer:-AGCAGCACGACTTCTTCAAGTC-3;reverseprimer:-TGTAGTTGTACTCCAGCTTGTGCC-3HumanprimarycellisolationandcultureconditionsBJfibroblastsandBJhTERTfibroblastswereagenerousgiftfromJerryShayandWoodringWrightandwerecul-turedaspreviouslydescribed[52].ThehEScelllineH9(WA09,XX,Passage30-35)wasculturedonfeeder-freefibronectincoatedplateswithmouseembryonicfibroblast(MEF)conditionedhumanEScellmedium.MEFsweremitomycin-cinactivatedandplatedinfibro-blastmedium,DulbeccosModifiedEagleMedium(DMEM)(Invitrogen),supplementedwith10%bovinegrowthserum(Hyclone,Logan,UT),2mML-glutamine,100IU/mLpenicillin,and100mg/mlstreptomycin.24-hoursafterattachment,themediumwasreplacedwithhEScompletemedium(77%DMEM:F12(Sigma),20%KnockoutSR(Invitrogen),1%Non-Essentialaminoa-cids(Invitrogen),1%Penicillin/Streptomycin(Invitro-gen),1mML-Glutamine(Invitrogen),0.1mMbeta-mercaptoethanol(Sigma),4ng/mlbasicFibroblastGrowthFactor(Invitrogen).After24hours,themediumwasremoved,filtered,andusedasconditionedmediumforhumanEScellcultures.Cellswereculturedin5%CO37°Candpassagedevery5-6daystomaintainundifferenti-atedcultures.HUVECcells(Lonza)wereagenerousgiftfromChiekoMineoandPhilShaul(UniversityofTexasSouthwesternMedicalCenterinDallas(UTSW))andwereculturedinEndothelialCellGrowthMedium-2(EGM-2)(Lonza)withtheEGM-2BulletKit(Lonza).Keratinocytes(Invitrogen)wereculturedinKeratinocyteSerumFreeMedia(KSFM)+supplement(Invitrogen).Thehema-topoieticprogenitorcells(Lonza)wereisolatedbyCD34+purificationfrombonemarrowandwereculturedinhematopoieticprogenitorcellmedium(Lonza).HumancelllinescultureconditionsCaCo-2cellswereagenerousgiftfromJerryShayandWoodringWright(UTSW)andwereculturedinDMEM(MediaTech)supplementedwith10%bovinegrowthserum(Hyclone,Logan,UT),2mML-glutamine,100IU/mLpenicillin,and100mg/mlstreptomycin.Theculturesweregrowninahumidifiedincubatorat37°Cwith5%.HBEC3KTcellswereagenerousgiftfromJohnMinna(UTSW)andwereculturedaspreviouslydescribed[53].HeLacellswereculturedinHepG2cells(ATCC)wereculturedinthesamewayasCaCo-2cells.HT-29cellswereagenerousgiftfromJerryShayandWoodringWright(UTSW)andwereculturedinthesamewayasCaCo-2cells.JurkatcellswereagenerousgiftfromZhijianChen(UTSW)andwereculturedinRoswellParkMemorialIn-stitute1640(RPMI)(MediaTech)supplementedwith10%bovinegrowthserum(Hyclone,Logan,UT),2mML-glutamine,100IU/mLpenicillin,and100mg/mlstrepto-mycin.Theculturesweregrowninahumidifiedincubatorat37°Cwith5%CO.K562(ATCC)cellswereculturedinthesamewayasJurkatcells.MCF-7cellswereagenerousgiftfromRolfBrekken(UTSW)andwereculturedinthesamewayasCaCo-2cells.TF1cellswereagenerousgiftfromSaswatiChatterjee(CityofHope)andwereculturedinthesamewayasJurkatcells.Saos-2cells(ATCC)wereculturedinthesamewayasCaCo-2cells.U2OScellswereagenerousgiftfromDavidSpector(ColdSpringHarbor)andwereculturedinthesamewayasCaCo-2cells.MouseprimarycellisolationandcultureconditionsAdultskinfibroblasts,astrocytes,EScells,andembry-onicfibroblastswereisolatedandculturedasdescribedpreviously[54].Thehematopoieticprogenitorcellswereisolatedfromsixtoeightweekoldmice.Miceweresacrificedandwholebonemarrowwasflushedfromfe-mursandtibiaswithIMDM2%FBS.Nextwholebonemarrowcellswerespundownandresuspendedinetal.VirologyJournalPage7of10http://www.virologyj.com/content/10/1/74 MACSbuffer.CD117+cellswereenrichedfromwholebonemarrowbyusingMACSmagneticbeadseparationwithCD117+microbeads(Miltenyi),runningcellsoveraMACsMS+column(Miltenyi),andwashingthecolumnthreetimeswithMACSbuffer.ThecolumnwasthenremovedfromthemagneticfieldandcellsretainedinthecolumnwereflushedwithMACSbufferusingaplunger.TheCD117+werethenwashedandlabeledwithantibodiestofurtherenrichforlong-termrepopulatingcellsusingthefollowingantibodies:non-specificbindingofantibodieswasblockedbyincubatingcellswithaCD16/32antibody(eBioscience),followedbylabelingcellswithLin-antibodiesFITC-CD3e(eBioscience),FITC-CD4(eBioscience),FITC-CD5(eBioscience),FITC-CD8a(eBioscience),FITC-CD11b(eBioscience),FITC-CD45R(eBioscience),FITC-Ly-6G(eBioscience),FITC-Ter119(eBioscience).AnAPC-Sca-1(eBioscience)antibodywasusedtolabelSca-1+cells.FITC-/APC+cells(KLScells)weresortedusingaFACsAriaflowcytometer(BDBio-science)andculturedinStemspan(StemcellTechnolo-gies).Murinekeratinocyteswereisolatedandculturedasdescribedpreviously[55].Themesenchymalstemcellswereisolatedandexpandedfrom8weekoldmiceaspreviouslydescribed[56].Briefly,wholebonemar-rowwasflushedfromfemursandtibiasandplatedinMesencultProliferationkitmedia(StemcellTechnolo-gies)insixwellplatesatadensityof5E6cellspermLandculturedasdescribedpreviously.Theskeletalmuscleprogenitorcellswereisolatedandculturedasdescribedpreviously[57](instructionintheisolationmethodwasgenerouslyprovidedbyAmyWagersatHarvardUniversity).Thewhiteadiposeprogenitorcellswereisolatedandculturedasdescribedpreviously[58](isolationinstructionwasgenerouslyprovidedbyJonGraffatUTSW).Mouse,hamster,andmonkeycelllinescultureconditions3T3cellswerecreatedaspreviouslydescribed[54]andwereculturedinthesamewayasCaCo-2cells.C2C12cellswereagenerousgiftfromEricOlson(UTSW)andwereculturedinthesamewayasCaCo-2cells.MIN6cellswereagenerousgiftfromMelanieCobb(UTSW)andwereculturedasdescribedpreviously[59].CHOcellswereagenerousgiftfromBenChen(UTSW)andwereculturedinthesamewayasCaCo-2cells.COS-7cellswereagenerousgiftfromCaroleMendelson(UTSW)andwereculturedinthesamewayasCaCo-2cells.AAVinfectionandmeasurementofGFPpositivecellsExperimentsforallcelltypeswereperformedinthisman-ner,unlessotherwisenoted.About10,000cellsperwellwereplatedin500Lofmediaina24-wellplate.Immedi-atelyafterplating,thecellswereinfectedwith1ofthe9AAVserotypesatamultiplicityofinfection(MOI)of100,000viralgenomespercell(theexperimentswerere-peatedusinganMOIof10,000,notshown).At24hours,anadditional0.5mLofmediawasaddedtocells(forthecellsina24wellplate,otherwisethevolumewasdoubledfromtheoriginalvolume).At48hourspostinfection,thecellswereharvestedandanalyzedforGFPexpressiononaFACSCaliber(Becton-Dickerson,SanJose,CA).ForhEScells,AAVtransductionexperimentswereperformed2daysafterpassageatatimewhenhEScellcolonieswereisolatedandpredominantlymonolayers.Formurinehematopoieticprogenitorcellsandmurinemesenchymalstemcells,cellswereplatedat10,000cellsperwellina96wellplateandinfected,asdescribedabove.Forwhitefatprogenitorcells,cellswereplatedat10,000cellsperwellina48wellplateandinfectedasdescribedabove.Formurinekeratinocytes,cellswereplatedat20,000cellsperwellina48wellplateandinfectedasdescribedabove.Formurineskeletalmuscleprogenitors,cellswereplatedat2,500cellsperwellina96wellplateandinfectedasdescribedabove.ManyoftheexperimentsatanMOIof100,000weredoneonce,someweredonetwoorthreetimes.Experi-mentswerethenrepeatedatanMOIof10,000andthetrendswerethesame(although,asexpected,the%GFPwerelower).Thus,quantitativevaluesoftransductionefficienciesoftheseserotypescannotbedeterminedbythisdata.However,becauseonlyonepreparationofeachserotypewasusedforallexperimentsforconsistency(andaliquotedandfrozenat-80°C),thisqualitativedatacanbeusedtochoosethebestserotype(s)fortransduc-tionofaparticularcelltype.Furthermore,theprepsofvirusmadeforthissurveywerefirstevaluatedon293cellstoensurethattransductionefficiencywascompar-abletothecountlessotherprepsthathavebeenmadeoftheseserotypes.AAV:Adeno-associatedvirus;eGFP:Enhancedgreenfluorescentprotein;PCR:Polymerasechainreaction;PBS:Phosphatebufferedsaline;HEK:Humanembryonickidney;MOI:Multiplicityofinfection;CMV:Cytomegalovirus;TALEN:Taleffectornuclease;ORF:Openreadingframe;scAAV:SelfcomplimentaryAAV;ssAAV:SinglestrandedAAV;HUVEC:Humanumbilicalveinendothelialcell;ES:Embryonicstem;hES:Humanembryonicstem;mES:Murineembryonicstem;hTERT:Humantelomerasereversetranscriptase;CD:Clusterofdifferentiation;MEF:Murineembryonicfibroblast;CHO:Chinesehamsterovary;MACS:Magneticactivatedcellsorting;FACS:Fluorescentactivatedcellsorting;vg:Viralgenomes.CompetinginterestsTheauthorshavedeclaredthatnocompetinginterestsexist.BEperformedinfections,isolatedprimarycells,performedflowcytometry,andwrotethemanuscript.JBperformedinfections,isolatedprimarycells,andperformedflowcytometry.MHmadetheAAVandhelpedwithdrafting/revisingthemanuscript.JCisolatedprimarycells.RScreatedtheheatmaps.MPconceivedthestudyandhelpedwithdrafting/revisingthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.informationBrianLEllis,MatthewLHirsch,JennyCBarkerm,denotesco-firstauthorship.etal.VirologyJournalPage8of10http://www.virologyj.com/content/10/1/74 AcknowledgementsWethankallofthepeoplementionedintheMaterialsandMethodssectionandtheirlabsfortheirgenerousgiftsofcellsandhelpwithisolation.WethankKelleyEllisandShainaPorterforherinsightfulcommentsandcarefulreviewofthemanuscript.FundingsourcesforBE,JB,JC,andMPwerefromLaurieKraussLacobFacultyScholarAwardtoMP,AmonCarterFoundation,andtheBurroughs-WellcomeFoundation.FudingforJBwasalsofromUTSouthwesternMedicalScientistTrainingProgram.Theyallprovidedsupportforsalaryandsupplies.FundingforMHwasfromNorthwestGenomeEngineeringConsortiumforsalaryandsupplies.AuthordetailsDepartmentofBiochemistry,UniversityofTexasSouthwesternMedicalCenter,Dallas,TX,USA.GeneTherapyCenter,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA.DepartmentofOphthalmology,UniversityofNorthCarolinaatChapelHill,ChapelHill,NC,USA.DepartmentofPharmacology,GreenCenterforSystemsBiology,SimmonsCancerCenter,UniversityofTexasSouthwesternMedicalCenter,Dallas,TX,USA.DepartmentofPediatrics,UniversityofTexasSouthwesternMedicalCenter,Dallas,TX75390-9148,USA.Received:12July2012Accepted:14February2013Published:6March20131.DayaS,BernsKI:Genetherapyusingadeno-associatedvirusvectors.ClinMicrobiolRev2008,21:583593.2.MichelfelderS,TrepelM:Adeno-associatedviralvectorsandtheirredirectiontocell-typespecificreceptors.AdvGenet3.SonntagF,SchmidtK,KleinschmidtJA:AviralassemblyfactorpromotesAAV2capsidformationinthenucleolus.ProcNatlAcadSciUSA4.FerrariFK,SamulskiT,ShenkT,SamulskiRJ:Second-strandsynthesisisarate-limitingstepforefficienttransductionbyrecombinantadeno-associatedvirusvectors.JVirol5.McCartyDM,MonahanPE,SamulskiRJ:Self-complementaryrecombinantadeno-associatedvirus(scAAV)vectorspromoteefficienttransductionindependentlyofDNAsynthesis.GeneTher6.SummerfordC,SamulskiRJ:Membrane-associatedheparansulfateproteoglycanisareceptorforadeno-associatedvirustype2virions.JVirol7.AsokanA,HamraJB,GovindasamyL,Agbandje-McKennaM,SamulskiRJ:Adeno-associatedvirustype2containsanintegrinalpha5beta1bindingdomainessentialforviralcellentry.JVirol8.KashiwakuraY,TamayoseK,IwabuchiK,HiraiY,ShimadaT,MatsumotoK,NakamuraT,WatanabeM,OshimiK,DaidaH:Hepatocytegrowthfactorreceptorisacoreceptorforadeno-associatedvirustype2infection.JVirol9.KurzederC,KoppoldB,SauerG,PabstS,KreienbergR,DeisslerH:promotesadeno-associatedvirustype2infectionofmammarycarcinomacellswithlowcellsurfaceexpressionofheparansulphateIntJMolMed10.SummerfordC,BartlettJS,SamulskiRJ:AlphaVbeta5integrin:aco-receptorforadeno-associatedvirustype2infection.NatMed1999,11.QingK,MahC,HansenJ,ZhouS,DwarkiV,SrivastavaA:Humanfibroblastgrowthfactorreceptor1isaco-receptorforinfectionbyadeno-associatedvirus2.NatMed12.AkacheB,GrimmD,PandeyK,YantSR,XuH,KayMA:The37/67-kilodaltonlamininreceptorisareceptorforadeno-associatedvirusserotypes8,2,3,and9.JVirol13.AkacheB,GrimmD,ShenX,FuessS,YantSR,GlazerDS,ParkJ,KayMA:two-hybridscreenidentifiescathepsinsBandLasuncoatingfactorsforadeno-associatedvirus2and8.MolTher14.KaludovN,BrownKE,WaltersRW,ZabnerJ,ChioriniJA:virusserotype4(AAV4)andAAV5bothrequiresialicacidbindingforhemagglutinationandefficienttransductionbutdifferinsialicacidlinkagespecificity.JVirol15.SeilerMP,MillerAD,ZabnerJ,HalbertCL:Adeno-associatedvirustypes5and6usedistinctreceptorsforcellentry.HumGeneTher16.WuZ,AsokanA,SamulskiRJ:Adeno-associatedvirusserotypes:vectortoolkitforhumangenetherapy.MolTher17.WuZ,MillerE,Agbandje-McKennaM,SamulskiRJ:Alpha2,3andalpha2,6N-linkedsialicacidsfacilitateefficientbindingandtransductionbyadeno-associatedvirustypes1and6.JVirol2006,80:90939103.18.DiPasqualeG,DavidsonBL,SteinCS,MartinsI,ScudieroD,MonksA,ChioriniJA:IdentificationofPDGFRasareceptorforAAV-5transduction.NatMed19.ZincarelliC,SoltysS,RengoG,RabinowitzJE:AnalysisofAAVserotypes1-9mediatedgeneexpressionandtropisminmiceaftersystemicMolTher20.EllisBL,HirschML,PorterSN,SamulskiRJ,PorteusMH:nuclease-mediatedgenecorrectionusingsingleAAVvectortransductionandenhancementbyFoodandDrugAdministration-approveddrugs.GeneTher21.PorteusMH,CathomenT,WeitzmanMD,BaltimoreD:Efficientgenetargetingmediatedbyadeno-associatedvirusandDNAdouble-strandMolCellBiol22.Smith-AricaJR,ThomsonAJ,AnsellR,ChioriniJ,DavidsonB,McWhirJ:Infectionefficiencyofhumanandmouseembryonicstemcellsusingadenoviralandadeno-associatedviralvectors.CloningStemCells2003,23.RabinowitzJE,BowlesDE,FaustSM,LedfordJG,CunninghamSE,SamulskiCross-dressingthevirion:thetranscapsidationofadeno-associatedvirusserotypesfunctionallydefinessubgroups.JVirol24.HauckB,XiaoW:Characterizationoftissuetropismdeterminantsofadeno-associatedvirustype1.JVirol25.BowlesDE,RabinowitzJE,SamulskiRJ:Markerrescueofadeno-associatedvirus(AAV)capsidmutants:anovelapproachforchimericAAVJVirol26.ChoiVW,McCartyDM,SamulskiRJ:AAVhybridserotypes:improvedvectorsforgenedelivery.CurrGeneTher27.WuZ,AsokanA,GriegerJC,GovindasamyL,Agbandje-McKennaM,SamulskiRJ:Singleaminoacidchangescaninfluencetiter,heparinbinding,andtissuetropismindifferentadeno-associatedvirusJVirol28.LiW,AsokanA,WuZ,VanDykeT,DiPrimioN,JohnsonJS,GovindaswamyL,Agbandje-McKennaM,LeichtleS,Redmon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