基因治疗.ppt

GeneTherapy Background CancerCCVD Cardio CerebrovascularDiseasesViralDiseaseGeneticDisease morethan2000diseasesCNS CentralNervousSystemDiseaseImmuneDiseasesDiabetes Humanbeingsfightagainstallkindofdiseases Background ChemicaldrugsSurgeryPhysicalTherapyTransplantationtherapyImmunotherapyRegenerativemedicine Differenttreatmentstothediseases Genetherapy Background Definition GenetherapyistheuseofDNAasadrugtotreatdiseasebydeliveringtherapeuticDNAintoapatient scells Genetherapy UsingDNAtoexpressfunctionalprotein UsingsiRNAorshRNAtoattenuateabnormalgeneexpressioninmRNAlevel Genomeeditingtocorrectmutantgenesequence ZFN TALEN CRISPR Cas9 Background Applications Genetherapycanbeappliedinmanydiseases suchascancer viraldisease geneticdisease etal Andmostlyappliabletothesingle genedisorders Genetherapy ThefirstapprovedgenetherapycaseintheUnitedStatestookplaceon14September1990 Therearemorethan2000clinicaltrialshavebeinglaunchedinthepastsevenyears Background Technicalproblem Barrierstothetarget DNA RNAisunstableinthebloodstream canbeimmunogenicanddoesnotreadilycrossmembranestoentercells Nuclease renalfiltrationPoorselectivityandinefficiencyofenrichmentinthetargetcellortissue Background Differentdeliverytools VirusVector Retrovirus Lentivirus Adenovirus Adeno associatedvirus AAV HerpesSimplexVirus HSV 1 etal Non virusmethod CyclodextrinPolymers Lipids Peptide Antibodies Aptamers andsmallmolecules Background Virusbasedtransduction WidelystudiedRNAvirusWidehostrangeInfectdividingcellandjustinfectonceIntegrateintohostgenomeLowImmunogenicLongexpressionperiodInsertionmutationbyrandomintegrationandoncogenicityCan tinfectnon dividingcellandlowvirustiter Retrovirus Background Virusbasedtransduction RNAvirusderivedfromHIVCaninfectnon dividingcellLowImmunogenicLongexpressionperiodInsertionmutationbyrandomintegrationandoncogenicityLowvirustiter Lentivirus Background Virusbasedtransduction DNAvirusHighvirustiter upto1014VP ml andhighinfectionefficiencyWidehostrangeandlargetransgenecapacity 37kb Infectdividingandnon dividingcellLowintegrationlevel existasepisomeinhostcellNoinsertionmutationbyrandomintegrationandoncogenicityShortexpressionperiod 5 20days ComplexprocedureandmanipulationPotentialimmunogenicandinflammatoryresponse JesseGelsinger 18yearsold diedfromsevereimmuneresponsecausedbyadeno virusbasedgenetherapyin1999 Adenovirus Background Virusbasedtransduction DNAviruswithoutpathogenicitySpecifichostrangeInfectdividingandnon dividingcellLowintegrationlevel existasepisomeinhostcellNoinsertionmutationbyrandomintegrationandoncogenicity site specificintegrateinto19chromosome LongexpressionperiodNoimmunogenicandinflammatoryresponseSmalltransgenecapacity 3kb Lowvirustiter 1012VP ml ComplexprocedureandmanipulationHostrangelimitation Adeno associatedvirus AAV Background Virusbasedtransduction DNAvirusHighinfectionefficiencyInfectdividingandnon dividingcellNeurotropicvirusLargetransgenecapacity upto 150kb LongexpressionperiodHighimmunogenicandinflammatoryresponseandnecrosis HerpesSimplexVirus 1 Background Virusbasedtransduction HSV AAVAd EBVHSV EBVAd AAVAd retrovirus Hybridvirusvector Background siRNA basedgenetherapy Background Non virusmethod Chemicalmodification ChemicalmodificationcanmaketheRNAberesistanttothenucleasecleavage Background Non virusmethod Cyclodextrinpolymernanoparticles CandeliverbothsiRNAandplasmidDNA Firstappliedin1999Targeted ligandLowtoxicityStericstabilizationNomeasuredinnateimmuneresponseswhenadministeredintravenously Background Non virusmethod Liposome CandeliverbothsiRNAandplasmidDNA ProtectentrappedoligonucleotidesfromnucleasedegradationandrenalclearancePromotecellularuptakeandendosomalescapeTheyincludetheuseofcationicorioniz ablelipids shieldinglipids cholesterolandtargetingligands Background Non virusmethod Conjugatedelivery Firstreportedin2007PEG shieldingeffectGalNAcligandwasessentialforbothuptakebyhepatocytesandinvivosilencingactivity Othertargetingligandshasbeenexplored includingpeptides antibodies smallmolecules glycans lectinsandnucleicacids DynamicPolyConjugates DPC 99 knockdownoflivergenesafterasingle0 2mgperkgdoseinnon humanprimates withtheeffectlastingnearly7weeks Background Non virusmethod Conjugatedelivery ASGPR onhepatocytes TriantennaryGalNAc siRNA BothsubcutaneousandintravenousadministrationofthisconjugaterevealedgreataccumulationofsiRNAintheliverandimprovedknockdownofthetargetgene Background Self assemblyofoligonucleotidenanoparticles Conjugatedelivery 3D DNAtetrahedra Background CentralNervousSystemDisease BBB BloodBrainBarrier Background HepaticCell Background HepaticCell hydrodynamicinjection PlasmidDNA 60 g andssDNAoligo 60 g suspendedin2mlsalinewereinjectedviathetailveinin5 7secondsinto8 10weeksoldFahmut mutmice HereditarytyrosinemiatypeI HTI Fumarylacetoacetatehydrolase FAH homozygousG Apointmutationofthelastnucleotideofexon8ofFah Fah5981SBmousemodel Treatmenteffect Treatmenteffect 33 5 3 3 0 40 0 12 Side effectanalysis Indelandoff targeteffect Side effectanalysis Indelandoff targeteffect Indelandoff targeteffect Side effectanalysis mismatch specificSurveyornucleaseassay Discussion TransientexpressionofCas9 sgRNAandaco injectedssDNAbynonviralhydrodynamicinjectionissufficie