recombinant adeno-associated virus (raav) cloning … · recombinant adeno-associated virus (raav)...
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RecombinantAdeno-AssociatedVirus(rAAV)
CloningandVirusPackagingServiceManual
Revision201603.02
©VigeneBiosciences2016
RESEARCHUSEONLY.
Notforuseindiagnosticprocedures
ThisproductshallbeusedbythepurchaserforinternalresearchpurposeonlyandredistributionisstrictlyprohibitedwithoutwrittenpermissionfromViGene
BiosciencesInc.
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TableofContent
CONTENTSANDSTORAGE..............................................................................................................3
Introduction....................................................................................................................................4
Whichviralvectortouse-viralvectorselectionguide...............................................................5
AAVserotypesandnativetropism-AAVselectionguide...........................................................6
AAVRelatedServiceDetails............................................................................................................6
AAVvectorcloningservices........................................................................................................6
SelectionsofrAAVvectorsfromViGeneBiosciencesInc.......................................................6
pAV-FHforgeneexpression...................................................................................................7
rAAVShRNAvectors...............................................................................................................7
AAVCustomcloningserviceswithpre-selectedpromoters...................................................8
FLEX-ONofCredependentinducibleexpression.................................................................10
AAVVirusPackagingServices...................................................................................................10
FinalProductsComponentsandQCStandards........................................................................10
ThepurityoftheAAVvirus.......................................................................................................10
Thevirustiter............................................................................................................................11
Recommendedprotocolforinvitrocelltransductionandinvivoanimalinjection.................11
Invitrocelltransduction...........................................................................................................11
Invivoanimaluse.........................................................................................................................13
FAQ...............................................................................................................................................13
BiosafetyConsiderations:.............................................................................................................16
LIMITEDPRODUCTWARRANTY....................................................................................................16
ORDERINGINFORMATIONANDTECHNICALSUPPORT.................................................................16
Ordering....................................................................................................................................16
TechnicalSupport.....................................................................................................................16
CONTENTSANDSTORAGE
AAVstocksaresuppliedinliquidformatindicatedtiter.ThestoragesolutionisPBSwith0.001%F68.Storeat-80°C.Ifdesired,aliquotviralstockuponarrival,andstorethosealiquotsat-80°Cfreezerimmediately.Dependentondifferentservicetypes,theproductsmaycontainthefollowingcomponents.
1. SmallscalecrudeAAV.500ulofrAAVat10^12-13GC/ml.*
2. LargescalepurifiedAAV.500ulofrAAVat10^13-14GC/ml.*
3. CustomerLargescalepurifiedAAV.Customamount(upto10^16GC)ofrAAVat10^13-14GC/ml.*
• GC/mlstandsforAAVgenomecopies/mlmeasuredbyreal-timeqPCRin
comparisonwithastandardreferenceplasmidwithknowngenomecopynumber.ForallthevirusproductsfromViGeneBiosciencesIncthetitervirusismeasuredasgenomecopies/ml.InthisManual,weuseGCinterchangeablywithvg(viralgenomes)andvp(viralparticles)asaunitforviraltiters.
AAVclonesaresuppliedin5ugDNAinTEbufferofspecifiedamountoutlinedintheCertificateofAnalysis(CoA).
DONOTFREEZEANDTHAWREPEATEDLY.
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IntroductionInViGeneBiosciences,RecombinantAdeno-associatedVirus(rAAV)ExpressionSystemsareutilizedindeliveringandexpressingshRNA,humanORF,CRISPRinvitroandinvivo.
Adeno-associatedvirus(AAV)isasmallsinglestrandDNAviruswhichinfectshumansandsomeotherprimatespecies.AAVisnotcurrentlyknowntocausediseaseandhasverymildimmuneresponse.Itcaninfectdividingandnon-dividingcells.FurtherremovaloftherepandcapfromthevectorhaseliminatedtheAAVintegrativecapacity.ThosefeaturesmakerAAVidealviralvectorforgenetherapy.Todate,rAAVvectorshavebeenusedinmanyclinicaltrialsingenetherapy,promisingresultshavebeenachievedfromPhase1andPhase2trialsincluding,CFTR,HemophiliaB,Arthritis,andParkinson’sdiseases.Figure1showsthecellentryandtraffickingofrAAV.
AfterrAAVgetsintothecells,itstaysstableasepisomalDNA.Expressionofgeneusuallypeaksin5to10daysandcanlastseveralweeksorevenmonthsinvivo.
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Whichviralvectortouse-viralvectorselectionguideWhencomparingthreemostpopularviralvectorsingenedelivery,youshouldtakefollowingconsiderationbeforeyouchooseadeno-associatedviralvector.
1. Doyouneedtransientorstablegeneexpression?
2. Doyouneedtotransducedividingornon-dividingcells?
3. Howimportantispotentialimmuneresponsefromyourtargetcell?
4. Howlargeisthegeneofinterest?
AAVscaninfectdividingandnon-dividingcells.Itislowoncytotoxicityandimmunogenicity,suitableforlongtermgeneexpressioninnon-dividingcellsandshorttermindividingcellswithrelativehighgenedeliveryefficiency.ButAAVvectorhaslimitedcloningcapacity,thespacebetweentwoITRsisonly4.9kb,soyourgeneshouldbe3.5kborless.Pleaserefertothistableforchoosingyourviralvectorsystem.
Adenovirus Adeno-associatedVirus(AAV)
Lentivirus
Genome dsDNA ssDNA ssRNA(+)Coat Naked Naked EnvelopedGenomesize 38-39kb 5kb 9kbInfection/tropism Dividingandnon-
dividingcellsDividingandnon-dividingcells
Dividingandnon-dividingcells
HostGenomeInteraction
Non-integrating Non-integrating Integrating
Transgeneexpression
Transient Potentiallonglasting
Longlasting
PackagingCapacity 7.5kb 4.5kb 6kbImmuneResponse High VeryLow LowRelativeViralTiter 10^11GC/ml
withoutpurification
10^7GC/mlwithoutconcentration
10^7GC/mlwithoutconcentration
RelativeTransductionEfficiency
100% 70% 70%
RelativeForeignGeneExpression
High Medium Medium
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AAVserotypesandnativetropism-AAVselectionguideSofarthereare11AAVserotypesdescribed,theyallhavedifferenttropismandcaninfectcellsfrommultiplediversetissuetypes.Tissuespecificityisdeterminedbythecapsidserotype.Toselecttherightserotypesiscriticalindeliveryofgeneintodifferentcellsortissues.ThefollowingtablelistsmostpopularrAAVserotypeandtheirtropism.
AAVSerotype
TissueTropism(Xindicatesrecommendedapplication)
Muscle Liver Lung Brain Retina Pancreas Kidney
AAV1 X
Neuronsandglialcells X X
AAV2
X
AAV5
Lungalveolarcells
Neuronsandglialcells X
AAV6 X
X
AAV7 X
Neurons X
AAV8 X X
Neurons X X
AAV9 X X X Neurons X X X
IfyoucannotfindenoughinformationtodecidewhichserotypeofrAAVorwhichvirusvectorworksbestforyoursystem,youcantryourGFP-VirusTestingKit,Cat#CT10001.
AAVRelatedServiceDetails
WeoffertwoservicesforAAVdevelopmentandproduction.ThefirstisAACvectorcloningservice;theotherisAAVpackagingservices.
AAVvectorcloningservices
SelectionsofrAAVvectorsfromViGeneBiosciencesInc.CurrentViGeneBiosciencesInc.offerspAV-FHvectorforgeneexpressionandfourvectorsforshRNAexpression.
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pAV-FHforgeneexpressionInmostofcase,ORFinsertsareclonedbetweenAsisIandMluIsites.InotherrarecasethecombinationofAsisI-RsrII,AsisI-NotIorAscI-MluIareusedinthecloning.PleasecheckourwebsiteortheCOAforspecificclones.InthepAV-FHvector,ORFisfusedwithaFlag/Histagatitscarboxylterminus.Thevectorcontainsanampicillinmarkerforbacterialselection.ViGene’spAV-FHvectorisamammalianORFexpressionvector,dualtagsofFlagandHiscouldbeusedtodetectandpurifyproteinsexpressedinmammaliancells.
rAAVShRNAvectors1. pAV-U6-GFP2. pAV-U6-RFP3. pAV-H1-GFP4. pAV-H1-RFP
4559bp
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ToexpressshRNAwithrAAV,ViGeneprovidesthechoicesofeitherU6orH1promotertodrivetheshRNAexpressionandGFPorRFPasmammalianexpressionmarker,shownbytheexamplemapofpAV-H1-RFP.
AAVCustomcloningserviceswithpre-selectedpromotersCMVisaverystrongandthemostcommonlyusedpromoterindrivinggeneexpressioninvitroandinvivo.CMVpromoterdrivesubiquitousgeneexpressioninmosttissueandcelltypes.Duetothemethylation/silencing,expressionbyCMVpromoterdecreasesinvivoafter10to20weeks.Forbettertissueorcelltypespecificgeneexpressionandforlongtermstrongandstableexpression,manydifferentpromotersareofferedbyViGeneBiosciences.
5020bp
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Cat.# Promoter Size Description
PM10001 ALB 2.4kb Liver specific 10 timer stronger than CMV after 10 weeks
PM10002 GFAP104 845bp Hybrid of EF1a and GFAP
PM10003 CAG 944bp Strong promoter, ubiquitous expression in vivo
PM10004 CamKIIa 1.2kb Specific expression in excitatory neurons in the neocortex and hippocampus
PM10005 EF1A 1.2kb Ubiquitous, weaker than CMV but better for in vivo
PM10006 CK1.3 1.1kb
PM10007 CK0.4 217bp Calcium/Calmodulin-dependent kinase II alpha
PM10008 GFAP 2.0kb Specific in astrocyte
PM10009 MBP 1.3kb Myelin basic protein promoter, efficient transduction of oligodendrocytes by adeno-associated virus type 8 vectors
PM10010 EFFS 253bp A short version EF1A
PM10011 TBG 460bp Homo sapiens serpin peptidase inhibitor, clade A
PM10012 aMHC 0.4kb Mouse myosin heavy chain alpha promoter
PM10013 cTNT 702bp Specifically transduce cardiomyocytes
PM10014 Synapsin 471bp Specific in neuron
PM10015 Mecp2 230bp Truncated Mcep2 neuron specific
PM10016 c-fos 1.7kb Activity-dependent promoter
PM10017 MCK 1.35kb Muscle creatine kinase promoter/enhancer
PM10018 UBC 1.1kb Ubiquitous, weaker than CMV but better for in vivo
PM10019 PGK 400bp Ubiquitous, weaker than CMV but better for in vivo
PM10020 Somatostat 1.2kb Restricting expression to GABAergic neuron
PM10021 Rpe65 700bp Retinal Pigment epithelium-specific expression in vivo and in vitro
PM10022 Insulin1 1.0kb Specific in beta- cells of the pancreas
PM10023 3Xenhancer McK 728bp Much stronger than CMV in muscle, inactive in nonmuscle cell lines
and mouse liver
PM10025 NSE 1.3kb Neuron-specific enolase promoter
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FLEX-ONofCredependentinducibleexpressionCombinetissuespecificpromotersandCredependentFLEX-Oninducibledesigncangetmorerestrictedtissuespecificandtemporalexpression.InFLEX-ONsystem,geneisinreverseorientationdownstreamofpromoter,andthegeneisflankedbytwooppositelyorientatedloxPs.IntheabsenceofCre,genecannotbeexpressedandinthepresenceofCre,geneexpressioncanbeturnonorinduced.
AAVVirusPackagingServices
FinalProductsComponentsandQCStandardsUnlessspecifiedotherwise,AAVvirusesareproducedfrom10^9Hek293cellsandarepurifiedbyIodixanolgradientultracentrifugation.Resultedvirusareconcentratedto400ulwithvirustiternolessthan10^13GC/ml.Thepurifiedvirusisgoodforinvivoanimalresearch.
VigeneBiosciencesInc.providestherAAVviruspackagingservicesinafewformats.
1. SmallsacletestingAAVpackagingservice.Inthisservice,rAAVispackagedusing10^07HEK293Tcells.Thevirusisincrudecelllysate,withoutanypurificationorconcentration.Thetiterisaround10^9-11GC/ml.
2. LargescalepurifiedrAAVpackagingservice.Inthisservice,rAAVispackagedusing2.5X10^8HEK293cells.VirusesarepurifiedbyIodixanolgradientultracentrifugation.Resultedvirusareconcentratedto400ulwithvirustiternolessthan10^13GC/ml.Thetiterisaround10^12-15GC/mldependentonvirusserotypeandthesizeofinsert.
3. CustomlargescalepurifiedrAAVservice.Thepurityandvirusaredeliveredperthespecification.ViGeneBiosciencescandeliverupto10^16GCwithvirustiternolessthan10^13GC/ml.
ThepurityoftheAAVvirus
TheAAVproteincomponentsareVR182kDa,VR272kDaandVR362kDa.SoagoodAAVpurificationshouldonlyshowthreemajorproteinbandswhenthevirusisanalyzebySDA-PAGE.Followingimageshowedtheproteincomponents
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inourpurifiedAAVvirus.WeguaranteethepurityoftheAAVvirusbasedonthespecificationthatwegivefordifferentservices.
Thevirustiter
Thevirustiterisdeterminedbytheviralgenomecopynumberin1mlsamplebyQ-PCRandcomparedtocopynumberstandardsamples.FollowingexampleisdatafortitteringAAV-GFPvirus.
Recommendedprotocolforinvitrocelltransductionandinvivoanimalinjection
InvitrocelltransductionDeterminetheMOI(multiplicityofinfections)
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• MOImeansMultiplicityofInfection.MOIequalsnumberofviralparticles(vp)percell.Inotherwords,andMOIof1meansinfectingwith1viralgenome(vg)percell.InViGeneBiosciencesourpackagingefficiencyis~100%.GC/mlstandsforAAVgenomecopies/mlmeasuredbyreal-timeqPCRincomparisonwithastandardreferenceplasmidwithknowngenomecopynumber.ForallthevirusproductsfromViGeneBiosciencesIncthetitervirusismeasuredasgenomecopies/ml.InthisManual,weuseGC,vgandvpinterchangeablyasaunitforviraltiters.
ForallthevirusproductsfromViGeneBiosciencesIncthetitervirusismeasuredasvirusparticlesorgenomecopies/ml.AlthoughmeasurementofvirustiterinGC/mlisreproducibleineverylab,therealinfectionunitscouldbeverydifferentwhenit’sestimatedindifferentexperiments.Thusbeforeyoudoyourexperiments,youhavetoestimatetheInfectionUnitsofvirusinyourexperiments.InViGeneBiosciencesInc,weusuallydoaten-foldserialdilutionofvirusstartingwith1ulviralstockandendingin10^8GC/ml.Addthedilutedthevirustoyourcells,2-3dayslater,basedonhowmanycellsbeeninfectedtocalculateyourinfectionunits.Thegoalistoget100%ofinfectionwithoutcausinganyundesiredeffects.Todeterminethisoptimalconcentrationofvirusforyourstudy,wesuggestyoutoconductpilottestinginyourcelllinebyusingreporterAAVlikeAAV-GFP(ViGeneBiosciences,catalognumber#CV10003throughCV10009).AfteryouknowtheinfectionunitsoftheviralstockanddecidetheMOIyouaregoingtouseinyourexperiments,dilutetheviralstockwithrightMOIfirst.
Removetheoriginalcellculturemedia,andaddtheaboveAAV-containingmediatocellculture.Belowisageneralguidelinefortheamountofmediaused:24-wellplate:0.2-0.3ml12-wellplate:0.5-0.8ml6-wellplate:2ml/well60mm-plate:3-4ml/plate10cm-plate:8-12ml/plateIncubatecellswiththevirus-containingmediaforatleast6-12hours.Youdon’thavetoexchangethevirus-containingmediaforfreshmedia,butyoucandoitafter6-12hours.Itmaytake3-7daysaftertheAAVinfectiontodetectthegeneover-expression.
Recommendedprotocolforinvitrocelltransduction
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1. ThawtheAAVvirusonice,andkeepitonicethroughoutthedurationoftheexperiment.
2. AAVinfectioniscelltypedependent.Somecelltypesexhibitlowtransductionefficiency,whileotherstransduceveryreadily.
WhendesigningAAVtransductionexperiments,itisrecommendedtousedifferentserotypesofareportervectorsuchanAAVexpressingGFP((ViGeneBiosciences,catalognumber#CV10003throughCV10009)todetermineoptimalserotypefortransductionofyourtissueorcellculture.
3. StartcelltransductionatMOIof10^4and10^6GC/cellwhencellsarereadilytransducible.WithsomecelllinesahigherMOImightbeneeded.Lookforthehighesttransductionwithminimalcelldeath.Withsomecelllines,hightransductionlevelscannotbeachieved.
4. UsetheminimumconcentrationofFBSthatthecellscanwithstandwhenperformingthetransduction.Forexample,HT1080cellsaremaintainedusingmediacontaining10%FBS.Transductionsareperformedusingmediacontaining2%FBS.
5. Usetheminimumamountofmedianecessarytocoverthesurfaceoftheplate.Forexample,transductionsareperformedin6-wellplates,1mlofmediaperwellisused.
6. Lookforexpressionat24h,48h,72hand96h,posttransduction.
Invivoanimaluse• Therecommendedtiterforinvivoanimalinjectionis10^11GCpergram(bodyweight).
• DilutetheviruswithPBStoachievetheappropriateGCnumber.
• Proceedtotheintravenousinjectionortailveininjectionorlocalizedtissueinjectionasdemonstratedbylabs(forreferencespleasevisithttp://www.vigenebio.com/delivery/AAV-Systems/).
FAQWhenshouldIuserAAVinmyexperiments?
rAAVcandelivergeneintodividingandnondividingcellsfortransientgeneexpression.Ithasbeenusedinvivoandinvitro.Refertoourtablewhenyouarenotsurewhichvirualvectorshouldyouchooseforyourexperiments.YoualsocanpurchaseViGene’s“GFPvirustestingkit”.Cat#CT10001totestinvitroorinvivo.
What'sthebiosafetyrequirementforusingAAV?
RecombinantAAVconstructs,inwhichthetransgenedoesnotencodeeitherapotentiallytumorigenicgeneproductoratoxinmoleculeandareproducedinthe
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absenceofahelperviruscanbehandledinBiosafetyLevel1(BSL-1)facility.OtherwiseitshouldbehandledasbiohazardousmaterialunderBiosafetyLevel2(BSL-2)containment.PleasecheckwithyourInstitutionalBiosafetyCommitteeorrelatedNIHwebsitefordetailedinformation,ifyouneedmoreinformation.
IsrecombinantAAVreplicationdeficient?
ForwildtypeAAV,replicationisatextremelylowefficiency,withoutthepresenceofhelpervirus,suchasadenovirus.Forrecombinantadeno-associatedvirusproducedthesedays,thereplicationandcapsidgenesareprovidedintrans(inpRep/Capplasmid),andonlythe2ITRsofAAVgenomeisleftandpackagedintovirion,whiletheadenovirusgenesrequiredareprovidedeitherprovidedbyadenovirusoranotherplasmid,thelikelihoodforarecombinantAAVtoreplicateistheoreticallyimpossible.Thisisinsimilarschemetolentiviralvectorsproducedthesedays.What’sthecloningcapacityforrecombinantAAVs?
AAVhasapackagingcapacityof~4.7Kb.SincethetwoITRsofAAVisabout0.2-0.3Kbintotal,theforeignDNAthatcouldbeintroducedbetweenthese2ITRsshouldbe<4.4Kb,whichismuchsmallerthanthatofrecombinantadenovirus(7.5Kb).Inaddition,whenthelengthofinsertedDNAbetweenthe2ITRsisclosethemaximalallowed,i.e.,4-4.4Kb,thepackagingefficiencydecreasessignificantly.Forinstance,forgeneover-expressionfromcDNA,sincetheCMV-poly(A)elementisabout1Kb,sothemaximalallowablecDNAlengthisabout3Kb.Inaddition,ifyouareinterestedinGFPco-expression(fromaseparateexpressioncassette),giventheadditionalCMV-EGFP-poly(A)isabout2Kb,sothemaximalcloningcapacityforGFPco-expressingsystemisabout1.0-1.2Kb.
Fordouble-strandedAAV(dsAAV),thecapacityisonlyhalfofthesingle-strandedAAV(ssAAV).HowmanyAAVSerotypesdoesViGeneBiosciencesOffer?
Thereare11differentAAVserotypeshavebeenreportedsofar.ViGeneBiosciencesprovidethepackagingservicesofAAVserotype1,2,5,6,7,8,9.Total7differentAAVserotypes.HowstableareAAVvectors?Howshouldtheybestored?
StabilitystudiescarriedoutinhouseandbysomecolleaguesshowthatpurifiedAAVvectorsarehighlystableattemperaturesof4Corless.Werecommendaliquotinguponreceiptandstoringat-80oC.Onceanaliquotisthaweditcanbestoredat4oCforshort-termstorage,e.g.,2-3weeks,withoutsignificantlossofbiologicalactivity.
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What'sthedifferencebetweenphysicalandgenomicparticles?
AAVgenomeparticlesrelatetotheviralparticlesthathavebeensuccessfullypackagedwiththegenometobedelivered.DuringtheAAVpackagingprocessmanyparticlesareformedlackingthegenomicDNA,whichlacktheabilitytotransducethecellstheycomeintocontactwithandarethereforenon-functional.AsaresearcheryouprobablywanttheconcentrationoffunctionalAAVparticles.
Whatdoesacustomerneedtoprovide?IfcustomerisgoingtoordertheclonefromVigeneBioscience,onlythegeneaccessionnumberfromNCBIorcatalognumberfromVigeneBioscienceisrequired.IfcustomerprovidesAAVvectorandusingpackagingservicesfromViGeneBiosciences,1mgDNAfrommaxi-prepattheconcentrationof1mg/mlshouldbeprovidedbycustomer.
HowlongdoestherAAVcloning,smallscaleandlargescaleAAVproductiontake?Ifthe1mgtransfectiongradequalityplasmidscanbesupplied,smallscaleandlargescaleAAVproductionwilltake1-2weeks.IfthecustomersrequiresVigenetoconductsubcloningandplasmidprepfortheAAVplasmid,theentireprocesscantake4-5weeks.
HowmuchAAVdoIneed?
• Celltransductiono StartcelltransductionatMOIof10^4and10^6GC/cellwhencellsarereadily
transducible.WithsomecelllinesahigherMOImightbeneeded.Lookforthehighesttransductionwithminimalcelldeath.Withsomecelllines,hightransductionlevelscannotbeachieved.
• Animalinjectiono Therecommendedtiterforinvivoanimalinjectionis10^11GCpermouseor
2X10^9GC/g(bodyweight).Forintravenousinjectionslargerquantityofvirusesmaybeneededincomparisontolocalinjections.
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BiosafetyConsiderations:FollowtherecommendedNIHguidelinesforallmaterialscontainingBSL-1organisms.
LIMITEDPRODUCTWARRANTYThiswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarrantiesofanykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesofmerchantabilityorfitnessforaparticularpurpose,areprovidedbyViGeneBiosciences.ViGeneBiosciencesshallhavenoliabilityforanydirect,indirect,consequential,orincidentaldamagesarisingoutoftheuse,theresultsofuse,ortheinabilitytousethisproduct.
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TechnicalSupport
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