recombinant adeno-associated virus (raav) cloning … · recombinant adeno-associated virus (raav)...

RecombinantAdeno-AssociatedVirus(rAAV)

CloningandVirusPackagingServiceManual

Revision201603.02

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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.


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.


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


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.


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


ToexpressshRNAwithrAAV,ViGeneprovidesthechoicesofeitherU6orH1promotertodrivetheshRNAexpressionandGFPorRFPasmammalianexpressionmarker,shownbytheexamplemapofpAV-H1-RFP.

AAVCustomcloningserviceswithpre-selectedpromotersCMVisaverystrongandthemostcommonlyusedpromoterindrivinggeneexpressioninvitroandinvivo.CMVpromoterdrivesubiquitousgeneexpressioninmosttissueandcelltypes.Duetothemethylation/silencing,expressionbyCMVpromoterdecreasesinvivoafter10to20weeks.Forbettertissueorcelltypespecificgeneexpressionandforlongtermstrongandstableexpression,manydifferentpromotersareofferedbyViGeneBiosciences.

5020bp


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


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


inourpurifiedAAVvirus.WeguaranteethepurityoftheAAVvirusbasedonthespecificationthatwegivefordifferentservices.

Thevirustiter

Thevirustiterisdeterminedbytheviralgenomecopynumberin1mlsamplebyQ-PCRandcomparedtocopynumberstandardsamples.FollowingexampleisdatafortitteringAAV-GFPvirus.

Recommendedprotocolforinvitrocelltransductionandinvivoanimalinjection

InvitrocelltransductionDeterminetheMOI(multiplicityofinfections)


• 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


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


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.


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.


BiosafetyConsiderations:FollowtherecommendedNIHguidelinesforallmaterialscontainingBSL-1organisms.

LIMITEDPRODUCTWARRANTYThiswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarrantiesofanykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesofmerchantabilityorfitnessforaparticularpurpose,areprovidedbyViGeneBiosciences.ViGeneBiosciencesshallhavenoliabilityforanydirect,indirect,consequential,orincidentaldamagesarisingoutoftheuse,theresultsofuse,ortheinabilitytousethisproduct.

ORDERINGINFORMATIONANDTECHNICALSUPPORT

Ordering• Email:[email protected]• TollFree(USA):1-800-485-5808• Telephone:301-251-6638• Fax:301-251-6110

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recombinant adeno-associated virus (raav) cloning … · recombinant adeno-associated virus (raav)...

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