Adeno-associated virus (AAV) Guide - Addgene

Of the commonly used viruses, AAV produces the lowest immune response, is non-pathogenic even in the wild-type state, and is thus thought to be the most ... Skiptomaincontent ScienceGuides AAVGuide Adeno-associatedVirus(AAV)Guide Components CommonUses ViralIntegration Serotypes Pseudotyping AAVVariants References AAVComponents Thesmall(4.8kb)ssDNAAAVgenomeconsistsoftwoopenreadingframes,RepandCap,flankedbytwo145baseinvertedterminalrepeats(ITRs).TheseITRsbasepairtoallowforsynthesisofthecomplementaryDNAstrand.RepandCaparetranslatedtoproducemultipledistinctproteins(Rep78,Rep68,Rep52,Rep40-requiredfortheAAVlifecycle;VP1,VP2,VP3-capsidproteins).WhenconstructinganAAVtransferplasmid,thetransgeneisplacedbetweenthetwoITRs,andRepandCaparesuppliedintrans. InadditiontoRepandCap,AAVrequiresahelperplasmidcontaininggenesfromadenovirus.Thesegenes(E4,E2aandVA)mediateAAVreplication.Thetransferplasmid,Rep/Cap,andthehelperplasmidaretransfectedintoHEK293cells,whichcontaintheadenovirusgeneE1+,toproduceinfectiousAAVparticles.Rep/Capandtheadenovirushelpergenesmayalsobecombinedintoasingleplasmid;theseparationofRepandCapshowninthefigureontherightfacilitatestheviralpseudotypingdiscussedbelow. OverviewofAAVPlasmidSystem CommonUsesofAAV AAViscommonlyusedinoptogeneticsexperiments.Thesevirusesarepreferredoverlentivirusesbecausetheyremainprimarilyepisomal,whilelentivirusesintegrateintothegenome.Thisisimportantbecauselocalchromatinstructureatthesiteofgenomeintegrationcanaffecttheexpressionoftransgenes,aswellastheexpressionofneighboringgenes.Theshortcodingsequencesofchannelrhodopsins,halorhodopsins,andotheroptogeneticgenesenablesthemtobepackagedinAAVs. ClickhereformoreinformationonoptogeneticsplasmidsavailablefromAddgene. ThepopularCRISPR/Cas9genomeeditingsystemhasbeenmodifiedforusewithAAV;thissystemrepresentsamajorstepforwardforinvivogenomeediting. FormoreinformationonAAV-CRISPR,pleaseseethisblogpost. BrowsethearticlefromRanetal.,2015fromtheZhangLabto findplasmidsoptimizedforuseinAAV,orcontainingStaphylococcusaureus(SaCas9) . AAVisalsoapromisingmethodforgenetherapy.Ofthecommonlyusedviruses,AAVproducesthelowestimmuneresponse,isnon-pathogeniceveninthewild-typestate,andisthusthoughttobethemostsuitablevirusfortherapeuticapplications.ClinicaltrialsusingAAVforvariousgenedeliveryapplicationsarecurrentlyunderway. ReturntoTop ViralIntegration Aspartofitslysogeniccycle,wild-typeAAVintegratesintothehostgenomeataspecificsite,AAVS1onhumanchromosome19.ThissiteisfavoredduetothepresenceofaRepbindingelement;however,randomintegrationsmayoccuratamuchlowerfrequency.Asareplication-incompetentvirus,AAVcannotenterthelyticcyclewithouthelp.Anothervirus,suchasadenovirusorherpessimplexvirus,oragenotoxicagentsuchasUVradiationorhydroxyurea,isnecessaryforlyticcycleactivation. WhenrecombinantAAV(rAAV)isusedforresearchpurposes,theRepproteinissuppliedintrans,eliminatingtheabilityofrAAVtointegrateintoitspreferredsiteofgenomicintegrationonhumanchromosome19,termedAAVS1.Instead,therAAVgenomeistypicallyprocessedintoadouble-strandedcircularepisomethroughdoublestrandedsynthesis.Theseepisomescanconcatemerize,producinghighmolecularweightstructuresthataremaintainedextrachromosomally.rAAVaremorelikelythanwild-typeAAVtointegrateatnon-homologoussitesinthegenome,anddosoatabouta0.1%frequency.Nonetheless,themajorityofrAAVparticlesarethoughttobemaintainedinepisomesorconcatemers. Episomesdifferprofoundlyfromviralparticlesproducedduringalyticcycle.rAAVepisomescandevelopchromatin-likeorganizationandpersistinnon-dividingcellsforaperiodofyearswithoutdamagingthehostcell.Incontrast,viralparticlesproducedduringalyticcyclearequicklyreleasedthroughcelllysis.Episomalstabilityenableslong-termtransgeneexpressioninnon-dividingcellsandisakeyadvantageofrAAV. AAVSerotypes ElevenserotypesofAAVhavethusfarbeenidentified,withthebestcharacterizedandmostcommonlyusedbeingAAV2.Theseserotypesdifferintheirtropism,orthetypesofcellstheyinfect,makingAAVaveryusefulsystemforpreferentiallytransducingspecificcelltypes.ThechartbelowgivesasummaryofthetropismofAAVserotypes,indicatingtheoptimalserotype(s)fortransductionofagivenorgan. Tissue OptimalSerotype CNS AAV1,AAV2,AAV4,AAV5,AAV8,AAV9 Heart AAV1,AAV8,AAV9 Kidney AAV2 Liver AAV7,AAV8,AAV9 Lung AAV4,AAV5,AAV6,AAV9 Pancreas AAV8 PhotoreceptorCells AAV2,AAV5,AAV8 RPE(RetinalPigmentEpithelium) AAV1,AAV2,AAV4,AAV5,AAV8 SkeletalMuscle AAV1,AAV6,AAV7,AAV8,AAV9 ReturntoTop AAVPseudotyping ResearchershavefurtherrefinedthetropismofAAVthroughpseudotyping,orthemixingofacapsidandgenomefromdifferentviralserotypes.Theseserotypesaredenotedusingaslash,sothatAAV2/5indicatesaviruscontainingthegenomeofserotype2packagedinthecapsidfromserotype5.Useofthesepseudotypedvirusescanimprovetransductionefficiency,aswellasaltertropism.Forexample,AAV2/5targetsneuronsthatarenotefficientlytransducedbyAAV2/2,andisdistributedmorewidelyinthebrain,indicatingimprovedtransductionefficiency.Manyofthesehybridviruseshavebeenwellcharacterizedandmaybepreferredoverstandardvirusesforinvivoapplications. Scientistshavealsoexperimentedwithhybridcapsidsderivedfrommultipledifferentserotypes,whichalsoalterviraltropism.OnecommonexampleisAAV-DJ,whichcontainsahybridcapsidderivedfromeightserotypes.AAV-DJdisplaysahighertransductionefficiencyinvitrothananywildtypeserotype;invivo,itdisplaysveryhighinfectivityacrossabroadrangeofcelltypes.ThemutantAAV-DJ8displaysthepropertiesofAAV-DJ,butwithenhancedbrainuptake. AAVVariants Asmentionedabove,AAVimprovementshaveincludedtheproductionofsyntheticcapsidsandthemixingofcapsids/ITRsfromdifferentAAVserotypestocreatehybridviruseswithnewproperties.Othersystemvariantsinclude: Self-complementaryAAV(scAAV):OnedownsideofAAVisitssingle-strandedDNAgenome.Becausethevirusdependsonthecell’sDNAreplicationmachinerytosynthesizethecomplementarystrand,transgeneexpressionmaybedelayed.Toovercomethisrate-limitingstep,scAAVcontainscomplementarysequencesthatarecapableofspontaneouslyannealing,uponinfection,whicheliminatestherequirementforhostcellDNAsynthesis.Unfortunately,thistechniquefurtherlimitsthepackagingcapacityofAAVto2.4kb. MethodstoIncreasePackagingCapacity:ToincreasethepackagingcapacityofAAV,alongertransgenemaybesplitbetweentwoAAVtransferplasmids,thefirstwitha3’splicedonorandthesecondwitha5’spliceacceptor.Whenthesevirusesco-infectacelltheyformconcatemers,aresplicedtogether,andthefull-lengthtransgenecanthenbeexpressed.Thismethodallowsforlongertransgeneexpression,butexpressionismuchlessefficientthanwithasingleAAVvirus(∼5%).Anothertechniqueforincreasingpackagingcapacitydependsonhomologousrecombination.Inthismethod,ageneisdividedbetweentwotransferplasmids,butwithsubstantialsequenceoverlap.Co-expressioninduceshomologousrecombinationandexpressionofthefull-lengthtransgeneatverylowefficiency(<1%ofwildtype).Ifeitherofthesemethodscanbemademoreefficient,theuseofAAVviruseswouldnolongerbelimitedtosmalltransgenes,enablingthedevelopmentoffurtherAAVapplications. ReturntoTop AdditionalResources AAVPlasmidsAvailableatAddgene BiosafetyGuide PartsofanAAVTransferPlasmidVideo WebReferences VectorBiolabs:IntroductiontoAAV UniversityofSouthCarolina'sViralVectorCoreAAVOverview Publications Viralserotypesandpseudotyping/genetherapy: Designergenedeliveryvectors:molecularengineeringandevolutionofadeno-associatedviralvectorsforenhancedgenetransfer.KwonI,SchafferDV.PharmRes.2008Mar;25(3):489-99.PubMed. Adeno-associatedvirusserotypes:vectortoolkitforhumangenetherapy.WuZ,AsokanA,SamulskiRJ.Mol.Ther.2006Sep;14(3):316-27.Epub2006Jul7.PubMed. RecombinantAAVviralvectorspseudotypedwithviralcapsidsfromserotypes1,2,and5displaydifferentialefficiencyandcelltropismafterdeliverytodifferentregionsofthecentralnervoussystem.BurgerC,GorbatyukOS,VelardoMJ,PedenCS,WilliamsP,ZolotukhinS,ReierPJ,MandelRJ,MuzyczkaN.Mol.Ther.2004Aug;10(2):302-17.PubMed. Fromvirusevolutiontovectorrevolution:useofnaturallyoccurringserotypesofadeno-associatedvirus(AAV)asnovelvectorsforhumangenetherapy.GrimmD,KayMA.CurrGeneTher.2003Aug;3(4):281-304.PubMed. Vectorgenomeintegration: Adeno-associatedvirusvectorintegration.DeyleDR,RussellDW.CurrOpinMolTher.2009Aug;11(4):442-447.PubMed. AAVvariants: Self-complementaryrecombinantadeno-associatedvirus(scAAV)vectorspromoteefficienttransductionindependentlyofDNAsynthesis.McCartyDM,MonahanPE,SamulskiRJ.GeneTher.2001Aug;8(16):1248-54.PubMed. ExpandingAAVpackagingcapacitywithtrans-splicingoroverlappingvectors:aquantitativecomparison.DuanD,YueY,EngelhardtJF.MolTher.2001Oct;4(4):383-91.PubMed. ReturntoTop