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viralvector韩晓Methodsofgenedelivery•ViralVectors:–Adenovirus–Retrovirus–Lentivirus–Adeno-associatedvirus(AAV)–Herpessimplexvirus(HSV)•Non-viralvectorbased–NakedDNA(plasmidDNA):injectionorgenegun–Liposomes(cationiclipids):mixwithgenes•Ex-vivo•InvivoWhyuseviralvectors•Virusareobligateintracellularparasites•VeryefficientattransferringviralDNAintohostcells•Specifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)•Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinsertedGenerationofviralvectorforgenetherapy•Replication-competentvirus•Replication-defectivevirus–Amplicon:doesn’tencodestructuralproteins–Can’treplicatebeyondthefirstcycleofinfection•Elementsneededtogenerateamplicon–TransferVector:plasmid(promoter,geneofinterest,ori,packagingsignal)–Packagingvector(cosmidorcelllines):providetheviralstructuralproteinsforpackagingoftransfervector–Helpervirus(packagingoftransfervector):deletedPackagingsignalsequenceAdeno-associatedvirusvectors•Non-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobases•Dependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)•AAV-2mostlyusedforvectorAdeno-AssociatedVirus(AAV)•SingleStrandedDNAVirus–ViralITRs&Rep/Cap–~5kbCapacity•Integrating/Concatameric–LongTermExpression•ComplexProduction&Purification–MultipDNA&/orHelperVirus–LowerTitreThanAdenovirus•SerotypeDifferencesInReceptors–AAV2HeparanSulphate–AAV5SialicAcidGenerationofadeno-associatedvirusvectorCharacteristicsofAAVvector•Advantages–Integrationandpersistentexpression–Noinsertionalmutagenesis–Infectingdividingandnondividingcells–Safe•Disadvantages–Sizelimitation,4.9kb–Lowtiterofvirus,lowlevelofgeneexpressionAdenoviralvectors•Non-envelopeddsDNA,36kilobases•Earlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)•Causesabenignrespiratoryinfectionsinhuman•Serotypes2and5arecommonlyusedasvectorsEarlygenerationsofadenoviralvector(replicationdefective)GutlessAdenoviralvector(Amplicon)Modificationofthetropismofadenovirusvector•AdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitous•ModifythefiberproteinCharacteristicsofadenoviralvector•Advantages–Hightiters–Bothdividingandnon-dividingcells–Widetissuetropism–Easilymodifytissuetropism•Disadvantages–Transientexpression(notgoodforgeneticdiseases)–Highlyimmunogenic–Hightitersofviruscanbetoxic–MoresuitableforcancerimmunotherapyRetroviralvector•Moloneymurineleukemiavirus(MuLV)•Generationofreplicationdefectiveretroviralvector–Transferplasmidvector:•Geneofinterest•Longterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscription•Primerbindingsite(PBS)(originofreplication)•RNApackagingsignal•Polypurinetract(importantforreplication)–Packagingvector•CelllinestablytransfectedwithplasmidconstructscontainingGag/polandEnvGenerationofretroviralvectorPseudotypedretroviralvectorCharacteristicsofretroviralvector•Advantages–Integration:permanentexpression–Pseudotypedvirus•Disadvantages–Onlyinfectingdividingcells–Insertionalmutagenesis(tumorformation)•Activateoncogenes•InhibittumorsuppressorgenesLentiviralvectors•Infectionofnon-dividingcells(hepatocytes,neurons)•HIV,ahumanlethalpathogen–Deleteaccessorygenes–Provideanenvelopefromanon-retrovirus(VSV)–Developvectorsfromlentivirusesofnon-humanpathogens•SIV,FIV,EIAVetcHerpesvirusvectors•Herpessimplexvirus1,milddiseaseinhuman,norisk•LineardsDNA,152kb,abouthalfofthetotal81genesarenon-essentialforvirusreplication•40-50kbofforeignDNAcanbeaccommodated•Neurotropicvirus,targettonervoussystem•ReplicationdefectiveampliconparticlesComparisonofdifferentviralvectorsViralvectortitersmanupilationofimmunogenicityinfectingoftropismnon-dividingcellsAdenovirus1011terrificveryhighyesRetrovirus107goodlowonlylentivirusHerpesvirus107notsogoodlowyesAAV107notsogoodlowyesGenetherapy•Genetherapy:–tocorrectageneticdefectbytransferringofafunctionalnormalcopyofthegeneintocells•Examplesofdiseasescausedbygeneticdefect–Ornithinetranscarbamylase(OTCdeficiency)–Hemophilia(bloodcoagulationfactorsVIIIorIX)–SCID(severecombinedimmunodeficiency)–Musculardystrophy–Cysticfibrosis–SicklecellanemiaApplicationofgenetherapy•Geneticdisorder(deficiency):OTC•Cancer–Geneticpredisposition–Mutationinoncogeneortumorsuppressorgene•Autoimmunitydiseases:rheumatoidarthritis–Deliveryofcounteractinggene•Diseasesinvolveseveralgenesandtheenvironmentalinteract:diabetesFactorstobeconsideredinGenetherapy•Howtodelivergenestospecificcells,tissueandwholeanimals?(methodsofdelivery)•Howmuchandhowlongtheintroducedgenewillbeexpressed?•Thesiteanddoseofgenedelivery•Isthereanyadverseimmunologicalconsequenceofbothdeliveryvehicle(Virus)andthegeneinanimals?Isthereanytoxiceffects?Deathof18-yearoldJesseGelsinger•Liverdisease:OTCdeficiency(geneticdisease)•UniversityofPennsylvania•Highdoseofadenoviralvector(E1andE4genesdeleted)carryingthenormalcopyofOTCgenewasadministered•Suspectedcauseofdeath-Toxicityofhightiteradenoviralvector-Highimmunogenicityofadenoviralvector(animmunerevolt)AcaseofleukemiainaSCIDchildtreatedwitharetrovir