遗传病的治疗

TreatmentofGeneticDisease7/15/20241ThecurrentstateoftreatmentofgeneticdiseasePreventiveMetabolicManipulationGeneProductReplacementCellorOrganTransplantationGeneTherapy7/15/20242TherapyofGeneticDisordersPreventiveTherapyPrenataldiagnosisPreimplantationdiagnosis

(invitrofertilization,testingofembryo&implantationofnormalembryo)Preventivescreeningfordiseaseonset7/15/20243Prenataldiagnosis7/15/20244Preimplantationdiagnosis7/15/20245PreventivescreeningSomegeneticchangesareassociatedwithanincreasedriskoffuturehealthproblems,suchascertainformsofcancer.Onewell-knownexampleisfamilialbreastcancerrelatedtomutationsintheBRCA1andBRCA2genes.Managementmayincludemorefrequentcancerscreeningorpreventive(prophylactic)surgerytoremovethetissuesathighestriskofbecomingcancerous.7/15/20246TherapyofGeneticDisordersMetabolicManipulationDietaryrestriction(LactoserestrictionforLactasedeficiency;phenylalaninerestrictionforphenylketonuria)DietarySupplementation(VitaminCforScurvy,BiotinforBiotinidasedeficiency,StarchforG-6-Pdeficiency)Chelationandenhancedexcretion(copperchelationforWilsonDisease)Metabolicinhibitors(allopurinolforgout,Statinsforhypercholesterolemia,)7/15/20247EffectsofDietarySupplementationTherapywithOralPharmacologicDosesofBiotinBeforeBiotintreatmentAfterBiotintreatment7/15/20248TherapyofGeneticDisordersGeneProductTherapyHormone,proteinorenzymereplacementHormonesupplementation:Hypothyroidism:thyroidCongenitaladrenalhyperplasia:cortisolGrowthhormoneHemophilia;clottingfactorsDiabetes:insulinEnzymereplacementBetaglucosidase:GauchersAlphaglucosidase:PompeAdenosinedeaminase(PEG):ADA-SCID7/15/20249ExamplesofCurrentEnzymeTherapyCurrentFDAapprovedenzymereplacementtherapy Adenosinedeaminasedeficiency(SCID)-SeverecombinedImmunodeficiencyNotargetingtocells,butremovalofmetabolitesfrom plasma

SeveralLysosomalStorageDisordersGeneticdeficiencyofLysosomalEnzymesTherapy:Targetingofdeficientenzymetolysosomes7/15/202410Adenosinedeaminasedeficiency:cellular&metabolicinteractionsdATPdATPnldeoxyadenosinelymphoidcells&RBCsothercells7/15/202411Adenosinedeaminasedeficiency:cellular&metabolicinteractions:EffectofenzymetherapydATPdeoxy

adenosinelymphoidcells&RBCsdeoxyinosineInjectionofPEGCalfAdenosineDeaminase7/15/202412LysosomalStorageDiseasesLysosomes:intracellularorganellescontaininghydrolyticenzymesthatdegrademacromolecules(recyclingand“garbagedisposal”forcells)LysosomalenzymesaretargetedtolysosomesbyinteractionwithMannose6PO4receptors

inthecellLysosomalenzymescanbetakenupintothecellfromplasmabyinteractionwithMannose6PO4receptorsoncellsurface7/15/202413

GaucherDisease Approved1991

FabryDisease Approved2001/03MucopolysaccharidosisI Approved2003MucopolysaccharidosisVI Approved2005MucopolysaccharidosisII Approved2006PompeDisease Approved2006Niemann-PickBDisease Phase1TrialENZYMEREPLACEMENTTHERAPYFORLYSOSOMALSTORAGEDISEASESDiseaseCurrentStatus7/15/202414TherapyofGeneticDisordersCellorOrganTransplantationCells Bonemarrow ImmunodeficiencyDisordersOrgans

Kidney FabryDisease Liver Tyrosinemia7/15/202415ThecurrentstateoftreatmentofgeneticdiseaseManygeneticdisorders

resultfromgenechangesthatarepresentinessentiallyeverycellinthebody.Asaresult,thesedisordersoftenaffectmanybodysystems,andmostcannotbecured.

However,approachesmaybeavailabletotreatormanagesomeoftheassociatedsignsandsymptoms.7/15/202416ThecurrentstateoftreatmentofgeneticdiseaseForinbornerrorsofmetabolism-Limitingcertainsubstancesinthedietcanhelppreventthebuildupofpotentiallytoxicsubstancesthatarenormallybrokendownbytheenzyme.-Insomecases,enzymereplacementtherapycanhelpcompensatefortheenzymeshortage.Thesetreatmentsareusedtomanageexistingsignsandsymptomsandmayhelppreventfuturecomplications.7/15/202417ThecurrentstateoftreatmentofgeneticdiseaseForothergeneticconditions,treatmentandmanagementstrategiesaredesignedtoimproveparticularsignsandsymptomsassociatedwiththedisorder.Forexample,ageneticdisorderassociatedwithaheartdefectmightbetreatedwithsurgerytorepairthedefectorwithahearttransplant.

Assicklecelldisease,cansometimesbetreatedwithabonemarrowtransplant.Bonemarrowtransplantationcanallowtheformationofnormalbloodcellsand,ifdoneearlyinlife,mayhelppreventepisodesofpainandotherfuturecomplications.7/15/202418ThecurrentstateoftreatmentofgeneticdiseaseGeneticdisordersmaycausesuchseverehealthproblemsthattheyareincompatiblewithlife.Inthemostseverecases,theseconditionsmaycauseamiscarriageofanaffectedembryoorfetus.Inothercases,affectedinfantsmaybestillbornordieshortlyafterbirth.Althoughfewtreatmentsareavailablefortheseseveregeneticconditions,healthprofessionalscanoftenprovidesupportivecare,suchaspainrelieformechanicalbreathingassistance,totheaffectedindividual.7/15/202419ThecurrentstateoftreatmentofgeneticdiseaseMosttreatmentstrategiesforgeneticdisordersdonotaltertheunderlyinggeneticmutation;however,afewdisordershavebeentreatedwithgenetherapy.Thisexperimentaltechniqueinvolveschangingaperson'sgenestopreventortreatadisease.Genetherapy,alongwithmanyothertreatmentandmanagementapproachesforgeneticconditions,areunderstudyinclinicaltrials.7/15/202420WHAT'SGENETHERAPYGenetherapyisthetherapeuticdeliveryofnucleicacidpolymersintoapatient'scellsasadrugtotreatdisease.Byaddingacorrectedcopyofadefectivegene,genetherapypromisestohelpdiseasedtissuesandorgansworkproperly.Thisapproachisdifferentfromtraditionaldrug-basedapproaches,whichmaytreatsymptomsbutnottheunderlyinggeneticproblems.Genetherapywasconceptualizedin1972.Thefirstattempt,albeitanunsuccessfulone,atgenetherapywasperformedbyMartinClineon10July1980.7/15/202421ChoosingCandidatesForGeneTherapyBetweenSeptember1990andJanuary2014some2,000clinicaltrialshadbeenconductedorapproved.Geneticdisorderscausedbymutationsinsinglegenestendtobegoodcandidatesforgenetherapy,whilediseasesinvolvingmanygenesandenvironmentalfactorstendtobepoorcandidates.Itisstillanexperimentalandemergingmedicaltechnologythathasseennewpromiseinthe2010safterextensivechallengesandsetbacksinthefirsttwodecadesofitsexistence.7/15/2024227/15/202423EssentialrequirementsofgenetherapyforanInheritedDisorder---ChoosingCandidatesForGeneTherapyIdentityofthemoleculardefectAfunctionalcopyofthegeneKnowledgeofthepathophysiologicalmechanismFavorablerisk-to-benefitratioAppropriateregulatoryFomponentsforthetransterredgeneAnappropriatetargetcellStrongevidenceofefficacyandsafetyRegulatoryapproval7/15/2024241.Doyouknowwhichgenesareinvolved?Ifyouplantotreatageneticflaw,youneedtoknowwhichgene(s)topursue.YoumustalsohaveaDNAcopyofthegeneavailableinyourlaboratory.7/15/2024252.Doyouunderstandthebiologyofthedisorder?Todesignthebestpossibleapproach,youneedtolearnallyoucanabouthowthegenefactorsintothedisorder.Forexample,whichtissuesthedisorderaffects,whatroletheproteinencodedbythegeneplayswithinthecellsofthattissue,andexactlyhowmutationsinthegeneaffecttheprotein'sfunction.Willaddinganormalcopyofthegenefixtheproblemintheaffectedtissue?Orcouldgettingridofthedefectivegenefixit?7/15/2024263.Canyoudeliverthegenetocellsoftheaffectedtissue?Theanswerwillcomefromseveralpiecesofinformation,includingthetissue'saccessibilityandmolecularsignatures.Targetcell:longhalf-lifeorgoodreplicativepotentialinvivobeaccessiblefordirectintroductionofgenecanbeculturedinvitroforexample:stemcells(bonemarrow);lymphocyte;endothelialcells,etc.7/15/202427ApproachestoGeneTherapyInthemoststraightforwardcases,genetherapyaddsafunctionalcopyofagenetocellsthathaveonlynon-functionalcopies.Buttherearetimeswhensimplyaddingaworkingcopyofthegenewon'tsolvetheproblem.Inthesecases,scientistshavehadtothinkoutsidetheboxtocomeupotherapproaches.7/15/202428ApproachestoGeneTherapyDominantnegative-Somemutationsingenesleadtotheproductionofadominant-negativeprotein.Adominant-negativeproteinmayblockanormalproteinfromdoingitsjob(foranexample,seePachyonychiacongenita).Inthiscase,addingafunctionalcopyofthegenewon'thelp,becausethedominant-negativeproteinwillstillbetherecausingproblems.7/15/202429ApproachestoGeneTherapyGain-of-function-Again-of-functionmutationmakesaproteinthatactsabnormally,causingproblemsallonitsown.Forexample,let'ssayasignalactivatesproteinX,whichthentellsthecelltostartgrowinganddividing.Again-of-functionmutationmaymakeproteinXactivatecellgrowthevenwhenthere'snosignal,leadingtocancer.7/15/202430ApproachestoGeneTherapyImproperregulation-Sometimesadisordercaninvolveaproteinthatisfunctioningasitshould—butthere'saproblemwithwhere,when,orhowmuchproteinisbeingmade.Theseareproblemsofgeneregulation:genesneedtobeturned"on"intherightplace,attherighttime,andtotherightlevel.7/15/202431GeneralConsiderationsforGeneTherapyInsertinganormalgeneintoanonspecificlocationwithinthegenometoreplaceanonfunctionalgene.Replacingamutatedgenethatcausesdiseasewithahealthycopyofthegene.Inactivating,or"knockingout,"amutatedgenethatisfunctioningimproperly.Introducinganewgeneintothebodytohelpfightadisease.7/15/2024327/15/2024337/15/2024347/15/202435TwoTypesofGeneTherapy-1SomaticcellInsomaticcellgenetherapy(SCGT),thetherapeuticgenesaretransferredintoanyofanycellotherthanagamete,germcell,gametocyteorundifferentiatedstemcell.Anysuchmodificationsaffecttheindividualpatientonly,andarenotinheritedbyoffspring.Somaticgenetherapyrepresentsmainstreambasicandclinicalresearch,inwhichtherapeuticDNA(eitherintegratedinthegenomeorasanexternalepisomeorplasmid)isusedtotreatdisease.7/15/202436TwoTypesofGeneTherapy-2GermlineIngermlinegenetherapy(GGT),germcells(spermoreggs)aremodifiedbytheintroductionoffunctionalgenesintotheirgenomes.Modifyingagermcellcausesalltheorganism'scellstocontainthemodifiedgene.Thechangeisthereforeheritableandpassedontolatergenerations.ThefirstgermlinegenetherapyconsistedofproducingageneticallyengineeredembryoinOctoberof1996.ThebabywasbornonJuly21,1997andwasproducedbytakingadonor'seggwithhealthymitochondria,removingitsnuclearDNAandfillingitwiththenuclearDNAofthebiologicalmother-aprocedureknownascytoplasmictransfer.7/15/2024377/15/202438MitochondrialAffectedfatherUnaffectedMotherUnaffectedfatherAffectedMotherUnaffectedchildrenAffectedchildrenUnaffectedAffected7/15/202439GeneTransferStrategiesThefirstinvolvesexvivo(i.e.,outsidethebody)introductionofthegeneintocellsthathavebeenculturedfromthepatientandthenreintroducedafterthegenetransfer.Inthesecondapproach,thegeneisinjecteddirectlyinvivointothetissueorextracellularfluidofinterest(fromwhichitisselectivelytakenupbythetargetcells).7/15/202440InvivoExvivo7/15/202441ThetargetcellsTheidealtargetcellsarestemcells(whichareself-replicating)orprogenitorcellswithsubstantialreplicationpotential.Atpresent,bonemarrowistheonlytissueforwhichstemcellsorprogenitorcellshavebeenusedsuccessfullyasrecipientsoftransferredgenes.Geneticallymodifiedbonemarrowstemcellshavebeenusedtocuretwoformsofseverecombinedimmunodeficiency,andotherdiseasesaffectingbloodcells,suchasthalassemiaandsicklecelldisease.7/15/202442SomaticTherapyforSCID

exvivoSevereCombinedImmunodeficiencyDisease(SCID)isduetoadefectivegeneforAdenosineDeaminase(ADA).Aretrovirus,whichiscapableoftransferringit'sDNAintonormaleukaryoticcells(transfection),isengineeredtocontainthenormalhumanADAgene.IsolatedT-cellstemlinecellsfromthepatientareexposedtotheretrovirusincellculture,andtakeuptheADAgene.Reimplantationofthetransgeniccellsintothepatient'sbonemarrowestablishesalineofcellswithfunctionalADA,whicheffecitvelytreatsSCID.7/15/202443ADAdeficiency(SCID):AshantideSilva,1990ThefirstapprovedgenetherapyintheUStookplaceon14September1990,attheNationalInstitutesofHealth(NIH),underthedirectionofWilliamFrenchAnderson.Four-year-oldAshantiDeSilvareceivedtreatmentforageneticdefectthatleftherwithADA-SCID,asevereimmunesystemdeficiency.Theeffectsweretemporary,butsuccessful.7/15/202444Stemcellbasedgenetherapy7/15/202445ClinicalTrialofStemCellGeneTherapy

forSickleCellDiseaseBoneMarrowHarvestIsolateStemCellsAddNormalHemoglobinGeneMyeloablatewithBusulfan(16mg/kg)TransplantGene-CorrectedStemCellsFreezeCertifyFollow:SafetyEfficacy7/15/2024467/15/2024477/15/2024487/15/2024497/15/2024507/15/2024517/15/2024527/15/2024537/15/2024547/15/202455GeneDelivery:ToolsOfTheTradeGenesaremadeofDNA.SuccessfulgenedeliveryrequiresanefficientwaytogettheDNAintocellsandtomakeitwork.ScientistsrefertotheseDNAdelivery"vehicles"asvectors.Thereisno"perfectvector"thatcantreateverydisorder.Likeanytypeofmedicaltreatment,agenetherapyvectormustbecustomizedtoaddresstheuniquefeaturesofthedisorder.Partofthechallengeingenetherapyischoosingthemostsuitablevectorfortreatingthedisorder.7/15/202456Tobesuccessful,avectormust:TARGET

therightcells.Ifyouwanttodeliverageneintocellsoftheliver,itshouldn'twindupinthebigtoe.INTEGRATEthegeneinthecells.Youneedtoensurethatthegeneintegratesinto,orbecomespartof,thehostcell'sgeneticmaterial,orthatthegenefindsanotherwaytosurviveinthenucleuswithoutbeingtrashed.ACTIVATEthegene.Agenemustgotothecell'snucleusandbe"turnedon,"meaningthatitistranscribedandtranslatedtomaketheproteinproductitencodes.Forgenedeliverytobesuccessful,theproteinmustfunctionproperly.AVOIDharmfulsideeffects.Anytimeyouputanunfamiliarbiologicalsubstanceintothebody,thereisariskthatitwillbetoxicorthatthebodywillmountanimmuneresponseagainstit.7/15/202457VectorsingenetherapyViralvectorsretrovirusadenoviruslentivirusherpessimplexvacciniaadeno-associatedvirusNon-viralnakedDNAelectroporationthegenegunsonoporationmagnetofection,oligonucleotideslipoplexesdendrimersinorganicnanoparticles7/15/2024587/15/2024597/15/202460Non-viralVectors7/15/202461CharacteristicsofviralvectorsAdvantagesofviralvectors:They'reverygoodattargetingandenteringcells.Sometargetspecifictypesofcells.Theycanbemodifiedsothattheycan'treplicateanddestroycells.Drawbacksofviralvectors:Theycancarryalimitedamountofgeneticmaterial.Therefore,somegenesmaybetoobigtofitintosomeviruses.Theycancauseimmuneresponsesinpatients,leadingtotwopotentialproblems:Patientsmaygetsick.Theimmunesystemmayblockthevirusfromdeliveringthegenetothepatient'scells,oritmaykillthecellsoncethegenehasbeendelivered.7/15/202462Vectorsusedingenetherapyclinicaltrials7/15/202463ConsiderationsWhatareyourtargetcells?Aretheydividingornon-dividing?Doyouwanttransientorlong-termexpression?Willanimmuneresponsetothevectoraffectyourresults?Isgeneexpressiongoingtobeevaluatedinvitroorinvivo?DoyouhaveaccesstoandtrainingforaBSL-2lab?7/15/202464TropismNotallviralvectorsare“designed”toinfectallcelltypes…tropismofvirus=tropismofvectorSomearemoreversatilethanothers–Retroviruses/lentivirusescanbepseudotypedwithawidevarietyofenvelopeproteinstobroadentropismMousespecific…ecotropicMuLVenvelopeMouseandhuman….amphotropicMuLVenvelopeMostvertebrates…VSV-G–AAVinherentlybroadtropism,butcanincreasewithdifferentserotypes–Adenovirusprettybroad,butneedsCAR(receptor)Lowinhumanhematopoieticcells&mousecellsingeneralCanswapfiberfromotherserotype7/15/202465PromoterissuesTissue/celltypespecificexpression–ie.Liver,Endothelialcells,CancercellsInducibleexpressionfortoxicgeneproducts–Tetracycline-operatorHighexpressionlevels–Viralpromoters(CMV;RSV;SV40)Downsideistheymaybetargetedbyanti-viralmechanismsinvivoConsistentexpressionlevels–Eukaryoticpromoters(PGK;EF-1)Multiplegenes–Muliplepromoters–Internalribosomeentrysite(ires)–Vectorsizerestraintsandvariableexpression–Self-cleaving2Apeptidefusedtotransgeneproduct•EspeciallyusefulforAAV(smallpackagingsize)7/15/202466RecombinantViralVectorSystemsVectorhascharacteristicsofparentvirus–Capsid/Envdictatestropism–Viralgenomemaintenancedictatestransientorlong-termexpression–Viralgenomesizedictatespackagingsize–ViralreleasedeterminesvectorpreparationCell-associatedand/orsupernatantSafetyFeatures–Packagingcelllines&helperconstructs7/15/202467ThestructureofadenovirusAdenoviruses(membersofthefamilyAdenoviridae)aremedium-sized(90–100nm),nonenveloped(withoutanouterlipidbilayer)viruseswithanicosahedralnucleocapsidcontainingadoublestrandedDNAgenome.Theirnamederivesfromtheirinitialisolationfromhumanadenoidsin19537/15/202468Anicosahedral,non-envelopedadenovirus(Ad)particleisshown7/15/202469SchematicdiagramoftheadenoviralgenomeAdenovirusgenomesarelinear,non-segmenteddouble-stranded(ds)DNAmoleculesthataretypically26-46Kbplong,containing23-46protein-codinggenes.7/15/202470Mapofadenovirusserotype5genomeanddifferentgenerationsofadenoviralvectors7/15/2024717/15/202472RecombinantAdenovirusAd5-basedvectorsrequireCoxsackie-adenovirusreceptor(CAR)ontargetcells–Lowinhumanhematopoieticcells&mousecellsingeneral•Transientgeneexpression•Highimmuneresponse•Hightiters&transductionefficiency7/15/202473RecombinantAdeno-associatedvirus(AAV)BroadtropismLowimmunogenicityHelpervirusfreesystemsBSL-1Disadvantageissmallpackagingsize4.5kbAdeno-associatedvirus(AAV)isasmallviruswhichinfectshumansandsomeotherprimatespecies.AAVisnotcurrentlyknowntocausedisease.Theviruscausesaverymildimmuneresponse,lendingfurthersupporttoitsapparentlackofpathogenicity.TheAAVgenomeisbuiltofsingle-strandeddeoxyribonucleicacid(ssDNA),eitherpositive-ornegative-sensed,whichisabout4.7kilobaselong.Thegenomecomprisesinvertedterminalrepeats(ITRs)atbothendsoftheDNAstrand,andtwoopenreadingframes(ORFs):repandcap.7/15/202474RetroviralvirioncomponentsGag=structuralproteins–Matrix–Capsid–Nucleocapsidpol=enzymes–Protease–ReverseTranscriptase–Integrase•Env=surfaceglycoproteins•Genome=RNARetroviridaeisafamilyofenvelopedvirusesthatreplicateinahostcellthroughtheprocessofreversetranscription.Virionsofretrovirusesconsistofenvelopedparticlesabout100nmindiameter.Thevirionsalsocontaintwoidenticalsingle-strandedRNAmolecules7–10kilobasesinlength7/15/202475Schematicrepresentationoftheretroviralgenome7/15/202476Retroviruscycle7/15/202477RecombinantRetrovirusesCanbepseudotypedwithvariousenvproteinstobroadentropismStablepackagingcellsLong-termgeneexpressionthroughintegration–DownsideisinsertionalmutagenesisDisadvantageisonlyinfectsdividingcells7/15/2024787/15/202479RetroviralvectordesignDeletepackagingsignalMaintainpackagingsignal7/15/202480TheProblemwithrecombinationRCR-replicationcompetentretrovirus7/15/202481TheSolutionSplittheHelpergenome7/15/202482Lentiviralvectors•Long-termgeneexpressionlikesimpleretrovirus•Advantageisabilitytoinfectnon-dividingcellsHIV-basedsystems•Non-humanbasedsystems•FelineImmunodeficiencyvirus(FIV)•Equineinfectiousanemiavirus(EIAV)•Simianimmunodeficiencyvirus(SIV)7/15/202483EvolutionofLentiviralVectorDevelopment7/15/202484VectorGeneticMaterialPackagingCapacityTropismInflammatoryPotentialVectorgenomeformsMainlimitationsMainadvantagesEnvelopedRetrovirusRNA8kbDividingcellsonlyLowIntegratedOnlytransducesdividingcells;IntegrationmightinduceoncogenesisinsomeapplicationsPersistentgenetransferindividingcellsLentivirusRNA8kbBroadLowIntegratedIntergrationmightinduceoncogenesisinsomeapplicationsPersistentgenetransferinmosttissuesHSV-1dsDNA40kb*150kb&StrongforneuronsHighEpisomalInglammatory;TransienttransgeneexpressionincellsotherthanneuronsLargepackagingcapacity;StrongtropismforneuronsNon-EnvelopedAAVssDNA<5kbBroad,withthepossibleexceptionofhaematopoieticcellsLowEpisomal(>90%);Integrated(<10%)SmallpackagingcapacityNon-inflammatory;Non-pathogenicAdenovirusdsDNA8kb*30kb$BroadHighEpisomalCapsidmediatesapotentinflammatoryresponseExtremelyefficienttransductionofmosttissuesThemaingroupsofviralvectors*Replicationdefective.&Amplicon.$Helperdependent.AAV,adeno-associatedviralvector;dsDNA,double-strandedDNA;HSV-1,herpessimplexvirus-1;ssDNA,single-strandedDNA.7/15/202485TargetedgenomeeditingtechniquesZFNTALENCRISPR/cas97/15/202486Zinc-fingernucleases(ZFNs)7/15/202487Transcriptionactivator-likeeffectornucleases(TALENs)7/15/202488CRISPR/Cassystem7/15/2024897/15/202490GenomeedittingingenetherapyZFNshavebeenappliedinthemonogenicdiseasessuchastheIL2Rγgeneandtheb-globingeneforgenecorrectionandCCR5geneformutagenesisanddisablement.TALENshavebeenutilizedexperimentallytocorrectgeneticerrorsunderlyingdisease.TALENshavebeenusedinvitrotocorrectthegeneticdefectsthatcausedisorderssuchassicklecelldisease,xerodermapigmentosum,andepidermolysisbullosa.CRISPR-Cas9-mediatedcorrectionofaFahmutationinhepatocytesinamousemodelofthehumandiseasehereditarytyrosinemia.7/15/202491ExamplesofZFN,TALEN,andCRISPR/Cas-mediatedgenomeediting

inhumancellsandmodelorganisms7/15/202492ZFNTALENCRISPRwhat'sit?AproteinconsistingofaDNA-cuttingenzymeandaDNA-grabbingregionthatcanbetailoredrecognizedifferentgenes.AlsoaproteincontainingaDNA-cuttingenzymeandaDNA-grabbingregionthatcanbeprogrammedtorecognizedifferentgenes.butitiseasiertodesignthanZFN.ADNA-cuttingproteinguidedbyanRNAmolecularthatisabletofindthespecificgeneofinterest.ProsandconsItwasthefirstprogrmmablegenome-editingtool,butitreliesonproteinsthatcanbedifficulttoengineerfornewgenetargets.potentiallydangerousoff-targetcutsarealsopossible.Thoughsimplerandcheaperthanzincfingernucleases.TALENproteinscanstillbedifficulttoproduceanddeliver.off-targetcutsareaproblem.Thistechniqueisaffordavleandeasytouse,anditworksforhigh-througout,multigeneexperiments.liketheothertools,itcanmakeoff-targetcuts.7/15/202493ApplicationofgenetherapyImmunedeficienciesHereditaryblindnessBlooddiseaseCancerHIV7/15/202494ImmunedeficienciesSeveralinheritedimmunedeficiencieshavebeentreatedsuccessfullywithgenetherapy.Mostcommonly,bloodstemcellsareremovedfrompatients,andretrovirusesareusedtodeliverworkingcopiesofthedefectivegenes.Afterthegeneshavebeendelivered,thestemcellsarereturnedtothepatient.Becausethecellsaretreatedoutsidethepatient'sbody,theviruswillinfectandtransferthegenetoonlythedesiredtargetcells.7/15/202495HereditaryblindnessGenetherapiesarebeingdevelopedtotreatseveraldifferenttypesofinheritedblindness—especiallydegenerativeforms,wherepatientsgraduallylosethelight-sensingcellsintheireyes.Encouragingresultsfromanimalmodels(especiallymouse,rat,anddog)showthatgenetherapyhasthepotentialtosloworevenreversevisionloss.7/15/2024967/15/202497HemophiliaPeoplewithhemophiliaaremissingproteinsthathelptheirbloodformclots.Thosewiththemost-severeformsofthediseasecanloselargeamountsofbloodthroughinternalbleedingorevenaminorcut.Inasmalltrial,researcherssuccessfullyusedanadeno-associatedviralvectortodeliverageneforFactorIX,themissingclottingprotein,tolivercells.Aftertreatment,mostofthepatientsmadeatleastsomeFactorIX,andtheyhadfewerbleedingincidents.7/15/202498ExcellentmodelforgenetherapyapproachesSinglegenedefect(deficiencyofFIXgene)Therapeuticgoalmodest;AnincreaseinplasmaFIXlevelsabove1%wouldbesufficientamelioratethebleedingphenotypeEfficacycanbeassessedbyvalidatedroutinelaboratoryassayTightregulationnotrequiredWiderangeofFIXlevelsarelikelytobeefficaciousandnon-toxic7/15/202499LowvectordosePlasmaFIXlevels,Subject1:2x1011vg/kgOffprophylaxisfor>48monthsbutnospontaneoushaemorrhageFIXconcusagedownby77%7/15/2024100

NobleedingepisodesoverpostgenetransferNotransaminitis;NoneedforsteroidsNotrequiredFIXconcentratesfollowinggenetransfer44Y:Missensemutation;antiAAV8IgG=6RU;ondemandX1week;16bleeds/year7/15/2024101BlooddiseaseIn2007,apatientreceivedgenetherapyforseverebeta-Thalassemia.Bloodstemcellsweretakenfromhisbonemarrowandtreatedwitharetrovirustotransfer