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Genetherapy

  Definition:deliveryandexpressionofgeneticmaterialleadingtoanalterationintheinstructionsetofacellforatherapeuticpurpose

  1960’s‐ideaofgenetherapyproposed

  1990‐firsthumangenetherapyclinicaltrial

  2010‐stillnogenetherapyproductcommerciallyavailable

  Somaticvsgerm‐linegenetherapy

Genetherapy‐makingitwork

How to deliver it? Where to deliver it?

How to control it?

What genetic material to deliver?

How to test it?

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Keygoalsforagenetherapystrategy

  Easytoadminister.

  Long‐termproductionofatherapeuticproteinortherapeuticeffectfollowingasingleapplication.

  Highlyspecific,targetedtodiseasecells/tissueonly,therebylittleornoside‐effects.

Whatgeneticmaterialtodeliver?  GenesandRNAinterferencesequences

  Correctingfaultsby:  Replacinganon‐functionalgenewithafunctionalcopy  Silencinganabnormallyfunctioninggene

  Alteringthephenotypeofacell

  Needtoknowsomethingaboutthediseaseprocess

  Inheritedoracquired,acuteorchronic

  Treatmentoutcome  Cure,alternaturalhistory,alleviatesymptoms

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Wheretodeliveryit?

During & Tipene-Hook,

New Ethicals 1997 May, 57-65

Factorstoconsider

  Manydiseasesaffectmultiplesites

  Effectsoncircuitrye.gbrain

  Genemayneedtobedeliveredtowidespreadareasorrestrictedtoaspecificorgan/groupofcells

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Gettinggenesintocells:genedelivery

  Exvivo‐genetransferperformedincellculturebeforetransplantation

  Invivo‐genetransferperformedinsitu

  Therapeuticgene‐transgene

  Successofgenetherapydependentonefficientgenetransfer

  Genetransfermediatedbygenedeliveryvehicles(=vectors)

www.biochem.arizona.edu/. ../Lecture25.html

Genedelivery‐non‐viralvectors

  PlasmidDNA  Liposomes

  DNAmixedwithcationiclipidsandpolymers(polylysine,protamine)becomesencasedwithinalipidbubble.

  Cellularuptakeviaanendocyticprocess.  Norestrictiononsizeofgene,easyand

cheaptomanufacture. Maindisadvantagesarerelativeinefficiency

ingenedeliveryandtransientexpression

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Genedelivery‐viralvectors

  Exploitsnaturalabilityofvirusestoinfectmammaliancells

  Geneticallyengineeredtocarryatransgenecassetteanddeposititwithinacell.

  Engineeredforasingleroundofhostcellinfectionbyremovalofviralgenesinvolvedinreplication.

VIRAL GENES

VIRAL PROTEINS

ASSEMBLY SPREAD OF INFECTION

VIRUS

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NO VIRAL GENES OR VIRAL PROTEINS

PROTEIN OF INTEREST

VECTOR

DISPLACED VIRAL GENES

GENE OF INTEREST

NO NEW VIRUS

Lifecycleofaviralvector

Engineeringvectorsfromviruses

  Molecularcloningtechniquesusedtogeneticallyengineeravirustocarryatransgenecassette  e.g.AAVrepandcapgenesareremovedand

replacedwithatransgenegenecassette

  Therapeuticgenesarecontrolledbypromoters,regulatoryelements

  Celllinesareusedtopackagevectorparticles

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AAVvectortransgenecassette

e.g AAV expression cassette

ITR rep cap ITRWild-type AAV

ITR transgene expression cassette ITR Recombinant AAV

145 1100 <1600 600 300 145bp

ITR promoter transgene RE pA ITR

ITR – AAV inverted terminal repeatRE – regulatory element, e.g. woodchuck postregulatory transcriptional element (WPRE)pA – polyA signal

 

Viralvectors‐types

  Retrovirus

  Herpessimplexvirus

  Adenovirus

  Adeno‐associatedvirus(AAV)

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  Retrovirus  7‐11kbssRNA;Followingtargetcellentry,RNAgenomeisreverse

transcribedintodsDNAwhichintegratesintothecellchromatin  Efficientintegrationoftransgeneintochromosomeleadingtostable

geneexpressionwhichpersistsinparentanddaughtercells.  Goodforexvivoapproaches.  Maindisadvantage‐potentialforrandomintegrationnearan

oncogene.Alsounabletoinfectnon‐dividingcellsasitrequiresonemitoticdivisionforintegrationandexpressionofthetransgene.

  Lentivirus(HIV)‐subclassofretroviruses,infectsbothdividingandnon‐dividingcells,stableandlong‐termgeneexpression.

  Adenovirus  36kbdsDNA

  Transducesnon‐dividinganddividingcells

  Hightitervectorproduced

  Maindisadvantage‐transienttransgeneexpressionduetoimmuneresponsesdirectedagainstadenoviralproteins;maybeovercomebyproducing“gutless”adenoviralvectors

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  Herpessimplexvirus(HSV1)  152kblineardsDNA  Broadhostcellrange  Maindisadvantage‐intrinsictoxicityandcontaminationofvector

stockswithwild‐type(pathogenic)virus.Difficultiesinmaintaininglong‐termgeneexpression

  Adeno‐associatedvirus(AAV)  4.7kbssDNA  >40serotypesisolatedfromhuman,primates  Non‐pathogenic  Transducesnon‐dividinganddividingcells  Long‐termgeneexpression(>2.5yrs)  Maindisadvantage‐smallsize

Howdoyouchoosetherightvectorforthejob?

  Dependentoncelltargets  Dividingvsnon‐dividingcells,e.gstemcellsand

post‐mitoticcellsdifficulttotransduce,rapidlydividingepithelialandcancercellseasytotransduce

  Transduction=vector‐mediatedgenetransfer

  Vectortropism=selectivityforacelltype  Dependentontheligandpresentonavectorand

whetherthetargetcellexpressestheappropriatecell‐surfacereceptor

  Geneinsertsize

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Achievingspecificity‐vectortargeting  Neededfor:

  Safety–minimisespotentialtoxicityofgeneproductinhealthycells  Increasesgenetransferefficiency–wanttominimiseamountsof

vectorneededtoproducegeneproductattherapeuticlevels

  Achievedby:  Modeofdeliverye.g.directinjectionintositeofinterest  Vectorselection

  Exploitormodifyspecificityofviralvectorsforcertaincelltypese.gAAVserotypes(human,primate)

  Pseudotypevectors  Modifycapsidshellthatallowsbindingtodifferentreceptors

eGFP expression in the hippocampus brain region following AAV2 vector or AAV1/2 vector-mediated gene transfer

AAV2 AAV1/2

Howtocontrolgeneexpression?

  Whyisitnecessary?

Kay et al. 2001. Nat. Med., 7, 33-40.

Figure 2. Transduction of the target cell.The vector particle containing the therapeutic gene sequences binds to a cell, generally through a receptor-mediated process and then enters the cell, allowing the genome to enter the nucleus. The vector genome may go through complex processes but ends up as dsDNA that, depending on the vector, can persist as an episome or become integrated into the host genome. Expression of the therapeutic gene follows.

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Geneexpression

  Leveloftransgeneexpressiondeterminedbypromoterchoiceandinclusionofothercis‐actingDNAelements.

  Aimistoachievestable,long‐termtransgeneexpression.

Recombinant protein

Gene therapy

Regulatingtransgeneexpression

  Essentialinmaintainingtransgeneproteinexpressionatsteadytherapeuticlevels

  Enablesflexibilityinadjustingdosageasdiseaseevolvesorfortherapiestailor‐madeforindividualpatients

  Builtinsafetymechanism

Clackson, Gene Therapy, 7, 120-125, 2000.

Gene therapy

Gene therapy - regulated

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Regulatorysystems  Endogenouspromoter‐regulatedtranscriptionsystemsthatareresponsiveto

physiologicalstimuli  e.g.glucoseresponsiveelement‐controlinsulinrelease

  Exogenousdrug‐regulatedtranscriptionsystem  e.g.Rapamycin,ecdysone,tetsystem

  Idealregulatorysystem  Lowbasalexpression  Beinducibletohighlevelsoverawidedoserangetoprovideausefuldose

responsiveness  Inductionshouldbeapositiveeffect‐i.eadministerdrugtoswitchiton  Drugshouldbeactivefollowingoraladministrationandhavenopleiotropic

effectsinmammaliancells  Regulatoryproteinshouldhavenoeffectsonendogenousgeneexpression

andbeofhumanorigintominimiseimmunogenicity

TetOn/Offregulatorysystem

  Transactivator‐regulatoryelement–expressesthetetracycline‐controlledtransactivator(rtTA)whichisachimericproteincomposedoftheTetrepressorfusedtotheVP16activationdomainofHSV.

  Tet‐responsiveelement(TRE)isupstreamofasilentpromoterdrivingthetransgeneofinterest.

Adapted from: http://www.bdbiosciences.com/clontech/tet/index.html

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Howtotestagenetherapystrategy?

  Testfunctionalityoftherapeuticgenecassettes

  Invitroandinvivoanimalmodelsofdisease.  Howadequatearethosemodelsanddotheyreflect

diseaseprocessesinhumans?

  Needmeasurableendpointstoassesswhethergenetherapyhastherapeuticeffectandbenefit

  Assaysforquantifyingand/orvisualisingtherapeuticproteinlevels

Currentstatusofgenetherapy  Toxicityofsomeviralvectorsystems

  Immuneresponsesdirectedagainstcellscontainingforeignproteins(viral)leadingtoeliminationofthetherapy.Thusthetherapymaybeshort‐lived.

  Enhancedimmuneresponsesagainstvectorsencounteredpreviouslymaymeanproblemsinre‐dosing.

  Potentialforsomevectorstorecoverabilitytocausedisease.

  Vectortargetingnotoptimised–transgenesexpressedinhealthyanddiseasedcells

  Safetymechanisms/controlofgeneexpressionnotsufficientlyoptimisedandlong‐termeffectsunknown

  Multigenedisorders  Bestcandidatesforgenetherapyarethosethatarisefrom

singlegenemutations.Manycommondiseases(e.gAlzheimer’s,heartdisease)involveeffectsonavarietyofgenes.

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Viralvectors:toolsforgenefunctionstudies

  Geneoverexpression  Expresspoorlycharacterisedgenestogainabetter

understandingoftheirphysiologicaleffects

  Generatinganimalmodelsofdiseasee.g.Parkinson’sHuntington’s,Alzheimer’sdiseases

  Geneknockout  RNAinterference

Advantagesovertransgenicanimals

  Canbeengineeredtoexpresssingleortwoforeigngenes,differentregulatoryelementsandpromoters

  Canbeadministeredatanydevelopmentalstage‐frominuterotoadulttosenescentanimals

  Eithershortorlong‐termCNSgeneexpression

  Expressioncanbeobtainedincrucialbrainregionswhilepossibleside‐effectsassociatedwithwidespreadoverexpressionofthegenecanbeavoided

  Nothost‐specific‐rats,primates,mice

  Inexpensiveandrapidtogeneratecomparedwithclassicaltransgenics

  Higherlevelsoftransgeneexpressionobtained

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Combineduseofgermlineandviraltransgenicmethods

  Canbecombinedwithtransgenicmousemodelsforstudiesofinteractionsofdisease‐causingproteinswithothercellularproteins

  Confirmgenespecificitywithtransgenicknockouts.Useviralvector‐mediatedgenetransferofthemissinggenetorescuethephenotype

  Observefurtherpotentiationordownregulationofgeneeffects

  Affectregulationofgenesindifferenttissuesorbrainregions

RatandprimatemodelsofParkinson’sdisease

  AAVvectorsusedtooverexpresswild‐typeandmutantformsofα‐synucleininthesubstantianigraofratsandprimates

  ShowmanyofthepathologicalfeaturesofPD

•  proteininclusions

•  dystrophicneurites

•  progressivelossofTHcellsintheSNpc

•  drug‐inducedrotationandmotordeficits

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Kirik D & Bjorklund A, Trends Neurosci. 26(7) 2003, 386-392

RNAinterference(RNAi)

  Aformofpost‐transcriptionalgenesilencinginwhichspecificsequencesofdouble‐strandedRNA(dsRNA)canbeusedtoknockdowntheexpressionofagenetarget

  Adaptedasatooltoinvestigategenefunction

  HistoryofRNAi

http://www.invitrogen.com /content.cfm?pageid=10088

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BiochemicalmechanismofRNAi

1.  dsRNAisintroducedintothecell

2.  DICERdigestsdsRNAinto~21bpdsDNA(short‐interferingRNAs;siRNAs)

3.  ThesiRNAsareintegratedintotheRNAInducedSilencingComplex(RISC)

4.  siRNAsundergostrandseparation.Theantisensestrandbindstoitscomplementary/targetmRNA

5.  Argonaute‐endonucleasewithintheRISCdegradesthetargetedmRNAhttp://www.ambion.com/techlib/tn/101/7.html

UsingRNAiasatoolformanipulatinggeneexpressioninmammaliancells

  Inmammaliancells,introductionoflongdsRNAinitiatesacellularinterferonresponsethatultimatelycausescellshutdownandleadstoapoptosis

  RNAicanbeachievedinmammaliancellsbydirectdeliveryof:

  syntheticshort‐interferingRNA(siRNA)   syntheticshort‐hairpinRNA(shRNA)  microRNA(miRNA)

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siRNA

  21‐23bpofdouble‐strandedRNAwitha3’dinucleotideoverhang

  AlgorithmsareusedtodesignRNAisequences(guidelinesprovidede.g30‐50%G/Ccontent).

  3‐5siRNAsequencescoveringthegeneofinterestareselectedandtestedforthedegreeofsuppressionofgeneexpression.

siRNA‐synthesis,delivery,effect

  Synthesisinvitro  Chemicalsynthesis  Invitrotranscriptionsystems

  Delivery  Cellculture‐transfection,electroporation  Invivomodels‐injectionordirect

application

  RNAieffect‐transient(3‐7days),partialtofullknockdownofgeneexpression

Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9.

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shRNA

Termination sequence • Pol III: string of 3-5 T’s • Minimal SV40 poly A signal

siRNA template • Hairpin siRNA • Sense and antisense strand of siRNA

Promoter/enhancer • Pol III, (U6, H1) • Pol II, (CMV)

shRNA

  Delivery

  Plasmids

  Viralvectors

  Effect

  Cellculture‐transientknockdown

  Invivo‐long‐term(>1week)effectsusingviralvectors

Davidson BL, Paulson HL. Lancet Neurol. 2004 Mar;3(3):145-9.

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MicroRNA(miRNA)

  EndogenousRNAipathwayinanimalcells

  Endogenous~21‐mersmallRNAmoleculesfromnon‐codingRNA(introns,independentmiRNAgenes)

  Regulategenetranscriptionbybindingtothe3’‐untranslatedregionsofspecificmRNAs

  Keyregulatorsofearlydevelopment,cellproliferation,celldeath,apoptosis,celldifferentiation,braindevelopment

  miRBASE‐http://microrna.sanger.ac.uk/  3000miRNAsequencesfromvariousspecies  SequencesofmanymiRNAsarehomologousbetweenspecies  800uniquemiRNAsinhumanswith400‐500conservedinmice  Regulateexpressionofatleast30%ofprotein‐codinggenes

miRNAprocessing

  Transcriptionofpri‐miRNA

  Processingintopre‐miRNAinthenucleusbyDrosha

  Exportofpre‐miRNAtothecytoplasm

  Dicercomplexprocessing

  miRNAstrandselectionbyRISC

http://www.ambion.com/techlib/resources/miRNA/mirna_pro.html

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miRNAreducesteadystateproteinlevels

  Translationalrepression  Imperfectduplex  Reducesproteinexpression

withoutimpactingoncorrespondingmRNAlevels.(mechanismstillunclear)

  mRNAdegradation  Perfectduplex

  Transcriptionalregulation  guidingchromatin

methylation

http://www.ambion.com/techlib/resources/miRNA/mirna_fun.html

UsingartificialmiRNAasatoolforgenefunctionstudies

  SimilartoapplicationsforsiRNA/shRNA

  Invitrogen‐BLOCK‐iT™system

https://catalog.invitrogen.com /index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12492

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PotentialApplicationsofRNAInterference

  Testinghypothesesofgenefunction

  Functionalscreeningandtargetidentification

  Targetvalidationfordrugdevelopment

  Potentiallynewtherapeuticapproachestotreatingdiseases‐anewapproachtoantisenseandnewpossibilitiesforgenetherapy

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GenetherapyforParkinson’sdisease

  Parkinson’sdisease  Progressiveneurodegenerativedisorderaffecting

~1%ofthepopulationovertheageof65

  Twotypes:sporadicandfamilial

  Clinicalsymptoms:disordersinmovement(restingtremor,rigidity,akinesia,bradykinesia).Non‐motorsymptomsincludedepressionanddementia.

Pathology

  Selectivedegenerationofdopaminergicneuronswithinthesubstantianigraparscompacta(SNpc)thatprojectaxonstothestriatum

  Lewybodies‐abnormalproteinaggregatesinthecytoplasmofneurons

  Dopaminedepletioninthestriatumcausesdisordersinmovement

  Focalpathologybuthasimpactonoverallbasalgangliacircuitry

Adapted from Lindvall & Bjorklund. Nat. Med. 2000. 6, 1207-1208

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Whatcausesthesedopaminecellstodie?

  Stilllargelyunclear.Possiblecontributors:  Oxidativestress  Mitochondrialabnormalities  Excitotoxicity  Disturbancesincalciumhomeostasis  Toxins

  Environmentale.gpesticides  Cellulare.g.dopamine,α‐synuclein

AnimalmodelsofPD

  Modelshavepredictivevaluewithregardtodopaminedeficiency

  Rodentmodels  6‐hydroxydopamine(6‐OHDA)injectedunilaterallyintothe

striatumorSNpc  MPTP(systemicinjection)‐MPTPconvertedtotoxicMPP+which

isselectivelytakenupbydopamineneurons,inhibitsmitochondrialrespiration

  Rotenone(chronicinfusionoflowdoses)‐mitochondrialcomplexIinhibitor

  transgenicmice(e.gα‐synuclein)Non‐humanprimate

  MPTP

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Treatmentstrategies

  Goals  Alleviatemotorsymptoms  PreventongoingcelldeathprocessintheSNpc

  Currentpharmacologicaltreatments  Focusonaugmentingstriataldopaminelevels

  L‐Dopa/carbidopa‐crossesthebloodbrainbarrierandisconvertedtodopaminebyaromaticaminoaciddecarboxylase(AADC)

  Dopamineagonistsand/ormonoamineoxidaseBinhibitorswhichpreventdopaminebreakdown,antioxidants,glutamateantagonistsmayprovidesomebenefits

  Problems  Lossofefficacyovertime,on‐offeffects,dyskinesias,

hallucinations  Donotaffectongoingcelldeathprocess

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GenetherapystrategiesforPD

  Threemainstrategies:  Biochemicalaugmentation‐alleviatingsymptoms

  correctdopaminedeficiency

  Neuroprotection‐alteringnaturalhistoryofthedisease  growthfactors(e.g.GDNF)

  Resettingbasalgangliacircuitry‐alleviatingsymptoms/preventingfurthercelldeath?  silenceoveractiveneuronalcircuitsbyexpressingglutamic

aciddecarboxylase(GAD)

Biochemicalaugmentation  Expressgenesinvolvedindopaminebiosynthesis

Tyrosine Tyrosinehydroxylase(TH),tetrahydrobiopterin

L‐Dopa

AADC dopamine

  Vectorsystemsused:HSV,Ad,AAV

  Maindisadvantageisinabilitytomaintaindopamineconcentrationsatappropriatetherapeuticlevel

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Neuroprotection

  Introducegrowthfactorgenesthatpromotecellsurvivaland/orregenerationofremainingneuronse.g.glial‐derivedneurotrophicfactor(GDNF),brain‐derivedneurotrophicfactor(BDNF),sonichedgehog

  UsefulinearlystagesofthediseasewhenthereisstillasignificantdopamineneuronpopulationintheSNpc

  GDNFgenetherapyinparticular,lookspromising‐improvementsinneurochemicalassessmentsandmotorsymptomsinrodentandprimatemodelsofPDusingAAVandlentiviralvectorsystems

GDNFpromotessurvivalandregeneration

  GDNFpromotesaxonregeneration(sprouting)andcorrectionofdopaminedeficiencyinstriatum

  Thiseffectonlyoccurredwhenthevectorwasinjecteddirectlyintothestriatum

Bjorklund et al., Brain Res., 886 (2000), 82-98

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Methodsusedtoassesswhethergenetherapyhastherapeuticeffectandbenefit

  Neurochemistry‐measuredopaminelevels(tissuepunches,microdialysis)

  Assaysforquantifyingand/orvisualisingtherapeuticproteinlevels‐e.gimmunohistochemistry,RT‐PCR,Westerns,ELISA

  Cellsurvival‐e.gTHasamarkerofdopamineneurons

  Non‐invasiveimaginge.g.PET,MRI

  Behavioural‐changesinmotorfunctionasafunctionalendpoint

Functionalassessmentinrodents

  Spontaneousmotoractivity

  Drug‐inducedmotoractivity‐rotationalbehaviour  Unilaterallesionsproducean

imbalanceinstriataldopaminelevelsbetweentherightandleftbrainhemispheresandupregulationofpostsynapticdopaminereceptorsand/orsignaltransductionsensitivityonthelesionedside.

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  Rateofrotation(e.gturns/min)isdirectlyproportionaltoseverityofthelesionanddopamineloss.

  Assessrotationbehaviourbeforeandaftertreatment.Expectationisthatifatherapyworks,willseeareductioninrotationrate.

Bjorklund et al., Brain Res., 886 (2000), 82-98

Bluesquares=AAVGDNFinstriatumRedsquares=AAVGDNFinSNGreen=AAVGDNFinSNandstriatumOpensquares=control

Resettingbraincircuitry

  Dopaminedeficiencyinstriatumhasconsequencesonoverallbasalgangliacircuitry

  Overactivityinthesubthalamicnucleus(STN)

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Beforedeepbrainstimulation

Afterdeep‐brainstimulation

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  UseageneticapproachtosilenceSTNneuronsbyintroducingGAD(glutamicaciddecarboxylase)‐enzymeinvolvedinGABAsynthesisinthesecells

  Principleissimilartodeepbrainstimulation,anestablishedtreatmentforPDinhumans

  Mayalsohaveaneuroprotectiveeffect

GADgenetherapyinhumans

  World’sfirstgenetherapytrialforPDapprovedbytheU.SFDAin2003

  Openlabel,safetyandtolerabilitytrialofAAV‐GADinjectedunilaterallyintoSTNof12patients

  FirstpatienttreatedAugust2003withsurgeryon12patientscompletedbyJuly2005

  1yearfollow‐upcompletedJuly2006

  Built‐insafetymechanism‐ablationoftheSTNisanestablishedtreatmentforPD.

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DeepbrainstimulationvsSTNgenetherapy

Studydesign

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Functionalassessmentinhumans‐GADgenetherapy

  Fluoro‐deoxyglucosePETimaging

  MRI

  Motorassessments‐UnifiedParkinson’sdiseaseratingscale(UPDRS),GlosserQOL

  Neuropsychologicalevaluation

  Bloodforhaematologyandchemistry,urinalysis,ECG

  BloodforantibodiestoAAVandGAD

GADgenetherapyledtoimprovementinoverallmovement(UPDRSscore)

“off “state “on” state

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Improvementinmovementontreatedside

FunctionalPETimaging

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NeuralnetworkactivitydiscriminatesPDandcontrols

IncreasedmetabolisminSMAafterGADgenetherapy

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CurrentstatusofPDgenetherapy

  Unravellingpathwaysinvolvedinmediatingdeathofdopaminergicneuronswillleadtoidentificationofnewtargets.

  Areastotarget:Ubiquitin‐proteosomalpathway(e.gparkin),heat‐shockproteins