基因轉移與基因治療

基因轉移與基因治療復旦大學遺傳學研究所薛京倫2003，101醫(yī)學ppt基因：染色體上的DNA片段，是遺傳信息結構和功能的基本單位?；驔Q定生老病死控制高矮胖瘦影響喜怒哀樂基因組：某一生物的細胞中所帶有的全部遺傳信息。2醫(yī)學ppt基因與疾病基因與人類的疾病密切相關：遺傳病是由于基因先天缺陷所致;腫瘤的發(fā)生涉及多種基因改變,包括癌基因激活或抑癌基因失活;高血壓,糖尿病等多基因病也涉及到多種基因的改變;由病原體所致的傳染病也和人體基因密切相關,存在易感人群和耐受人群.3醫(yī)學ppt

一、基因治療概念

基因治療(GeneTherapy)指將正常基因或有治療作用的基因通過一定方式導入靶細胞,糾正基因的缺陷或者發(fā)揮治療作用，從而達到治療疾病目的的生物醫(yī)學高技術。

4醫(yī)學pptTheapplicationofgeneticprinciplesinthetreatmentofhumandiseaseByintroductionofgenetic

materialintotargetcellsinordertocounteracttheeffectofadiseasegeneorintroduceanewfunctionSomaticandGermlineapproachespossible5醫(yī)學ppt基因治療的必要條件UnderstandingofthediseaseprocessStructure/functionofgenetobeintroducedEfficientdeliveryofgenecontrolofgeneexpressionPrevention/controlofimmuneresponsesAnimalmodelandassessmentoffunctionClinicaltrial6醫(yī)學ppt研究基因治療的三個基本步驟

尋找適當的靶細胞

導入目的基因

基因表達7醫(yī)學ppt體細胞基因治療始終需要考慮的問題

基因轉移的效率

治療的特異性

基因表達的持久性及其調節(jié)

治療的毒副作用

什么疾病

什么基因

什么載體

什么靶器官和靶細胞

什么基因導入方法8醫(yī)學ppt體細胞基因治療 將基因作為一種特殊“藥物”，通過體細胞基因轉移治療疾病 慢性治療 急性治療 預防 遺傳性疾病 獲得性疾病 功能喪失 功能獲得9醫(yī)學ppt二、基因治療和傳統(tǒng)的基因工程的區(qū)別

二者都著眼于尋找可治病或有其他應用價值的“目的基因”。基因工程：目的基因——載體——導入大腸桿菌、酵母和哺乳動物細胞——體外表達所需要的蛋白——經過分離純化獲得能用于治療或其他用途的蛋白純品,最終是制造出一種蛋白類的藥物。10醫(yī)學ppt基因治療——目的基因——載體——導入人體，目的基因在人體內的細胞中制造所需要的蛋白——達到治病的目的。基因治療在技術上一旦成功,其優(yōu)勢：①制品為基因及其載體，非基因表達蛋白產物,不需復雜的蛋白產物分離和純化工藝②生產成本遠遠低于基因工程產品③從理論上講,凡能治病的基因，都有可能開發(fā)成為“藥物”④半衰期11醫(yī)學ppt但基因治療難度高，技術要求極為苛刻。例如，針對各種疾病，必須具有能夠達到治病目的的基因。在此基礎上，還必須具有能有效地將基因導入人體的載體系統(tǒng)，這種系統(tǒng)要求高效，而且能定向地導入人體某種細胞?；驅肴梭w后，必須能夠控制它的表達。12醫(yī)學ppt因此，基因治療是生物高技術的高度集成，是遺傳學、分子生物學、細胞生物學、分子病毒學等多種學科知識和技術的高度綜合。

13醫(yī)學ppt三、基因治療的主要策略

GenereplacementGeneaugmentationtherapy(GAT)Genecorrection(Chimeraplasty)TargetedkillingofspecificcellsTargetedinhibitionofgeneexpression(Geneablation)14醫(yī)學pptGenereplacement:

Deficientgenecorrectedbyreplacingthemutatedallelewithanintactallele;usedforthetreatmentofautosomaldominantdisorders.Thestrategiesareasfollows:a)kockoutmutationbysinglecrossing-over(insertionalinactivation)

b)genereplacementbydouble(reciprocal)crossing-over

c)targetgeneinhibitionindominantnegativegeneticdisorders;usuallymutatedproteinswhichinteractwithnormalcellularproteinstocreatealteredproperties;mutationalinactivationoftheonemutatedcopywillalleviatethismutantphenotype;themutationcanbecarriedoutbyinsertionalinactivation,orantisensetechnology.15醫(yī)學pptGeneAugmentationTherapy(GAT)

FordiseasescausedbylossofgenefunctionmorecopiesofnormalgeneraiselevelsofgeneproductrestorenormalphenotypeApplyto:monogenicrecessivediseasescysticfibrosis,haemophilia,musculardystrophy16醫(yī)學pptGeneCorrection-Chimeraplasty17醫(yī)學ppttumourcelltkthymidinekinasegeneviralvectorganciclovirganciclovirphosphateTargetedKilling–GeneticPro-drugActivationTherapy18醫(yī)學pptTargetedinhibitionofgeneexpression

Ribozymes

-cancleave(orrepair)mRNATriplehelixoligonucleotides-blockgenetranscription

Antisense

oligos-blockmRNAtranslation19醫(yī)學pptAllgenetherapystrategiesdependongettingthegeneorgeneticmaterialintothetargetcells!!!

20醫(yī)學ppt四、基因轉移的主要方法：

若干相關術語：

Transfection轉染

：過去：將病毒DNA或RNA

轉入細胞。

現在：將外源DNA轉入動物細胞。

DNATransfer轉移：基因導入或轉移廣義的概念。

Transduction轉導：噬菌體介導的細菌之間的基因

轉移。

Transformation轉化：將裸DNA或質粒導入原核

和真核細胞。21醫(yī)學pptTwomainroutesofgenetransfer:Invivo: i.v.ori.m.injectable;or non-invasive(eg“sniffable”)Exvivo: hepatocytes,skinfibroblasts haematopoietic cells “bioreactors”22醫(yī)學pptaaaaaaEx-vivoIn-vivotopicaldeliveryIn-vivosystemicdeliveryVExamples:-bonemarrow-livercells-skincellsExamples:-brain-muscle-eye-joints-tumorsExamples:-intravenous-intra-arterial-intra-peritonealTHREEclassesofanatomicalgenedelivery23醫(yī)學ppt基因治療過程中的基因轉移方法

Viralvectors

Non-viralvectors

Physicalmethods

目前最有效的是病毒載體，但存在插入大小有限、免疫原性強和生產難等局限24醫(yī)學ppt1、病毒載體類型反轉錄病毒（RV）載體腺病毒（AV）載體腺相關病毒（AAV）載體單純皰疹病毒（HSV）載體慢病毒（lentivirus)載體等25醫(yī)學ppt對載體的基本要求：特異并有效的基因轉移特異、高效、持續(xù)性表達，具有可控性免疫原性低易于生產26醫(yī)學ppt載體特征：

reproducibility stablepropagated purifiedtohightiters mediatedtargeteddelivery27醫(yī)學pptAdvantageanddisadvantageofgene-transfervector(1)

Vector AdvantageDisadvantage

AVVeryhightransfectionexvivorepeatdosingineffectiveowingto

&invivostrongimmuneresponse

Transfectsproliferating&non-Insert-sizelimit0f7.5kb

proliferatingcells,substantialmanufacture,storage,QCare

clinicalexperienceacquiredmoderatelydifficult

efficientretargetedtransfctionshoutdurationofexpression

RVfairlyprolongedexpressionlowertransfectionefficiencyinvivo

hightransfectionefficiencyexvivoinsert-sizelimitof8kb

substantialclinicalexperienceexvivotransfectonlyproliferatingcells,safty

lowimmunogenicityconcernofinsertionalmutagenesis

Lentivirustransfectsproliferating&non-safetyconcernfromimmunodeficiency

proliferating,haematopoieticvirusorigins,manufacturing,storage,

stemcellsQCareextremelydifficult,insert-size

limitof8kb,noclinicalexperience

AAVefficientlytransfectsawidevarietyinsert-sizelimits4.5kb

0fcellsinvivo,veryprolongedmanufacture,QCareverydifficult

expressioninvivolittleclinicalexperience,safetyconcern

lowimmunogenicityofinsertionalmutagenesis,repeat

dosingaffectedlybyneutralizing

antibodyresponses28醫(yī)學ppt基因治療病毒載體比較

RVAVAAVHSVLV基因組成ssRNAdsDNAssDNAdsDNAdsRNA基因組長度10kb36kb5kb152kb10kb裝載容量<8kb8kb4.5kb30kb9kb病毒滴度107

1011

108

108108感染細胞譜窄寬寬窄寬感染能力中很強強強中整合能力有無有無強毒性作用遺傳毒細胞毒遺傳毒細胞毒遺傳毒免疫原性弱中等弱強弱29醫(yī)學pptAdvantageanddisadvantageofgene-transfervector(2)

Vector AdvantageDisadvantage

NakedDNAmanufacturing,storage,QCveryshortdurationofexpressionin

aresimpleandcheap,verymosttissues,veryineffecient

lowimmunogenicity,clinicaltransfectionexvivoandinvivo

limbischaemia,goodsafetyretargetingtransfectionvery

profiledifficult

Cationiclipidsrelativesimplemanufacturing,inefficienttransfectioninvivo

storage,QCefficienttransfectionveryshortdurationofexpression

exvivo,lowimmunogenicitylittleclinicalexperience

condensedrelativesimplemanufacturing,inefficienttransfectioninvivo

DNAparticlesstorage,QC.Efficienttrancfectionveryshortdurationofexpression

exvivo,lowimmunogenicitynowclinicexperience

goodsafetyprofile

retargetedtransfectiondemostraded30醫(yī)學pptaaaaaaTransfectionInfectionexposedto106particles/cell12hoursexposedto1particle/cell30minvirallymediatedgenetransferismillionsoftimesmoreefficentthannonviraltransfer(whencalculatedintermsoftransfer/particle)TransfectionversusInfection31醫(yī)學pptVectorusedingenetherapyTypeapproximateintegrationdurationofinsert-sizecapacitypersistenceNucleic-acidbased

Oligonucleotides:Decoys,antisense,Ribozomes,siRNA10-100kbnohours-daysExpressionplasmids2-10kbextremelyraredaysTransposons2-10kbefficientstablerelativerandomBacteriohageintegrase2-10kbefficient,siterestrictedstableArtificialchromosome50-300kbepisomal+/-stableVirusbasedRV2-6kbefficientstablerelativerandomLentiviral2-10kbefficientstablerelativerandomAAV2-5kbraremonths-yearsAV2-30kbNOweeks-monthsHerpesvirus2-40kbepisomalmoths-yearsEBvirus2-40kbepisomalmoths-yearsSV401-5kbmoderateunproven32醫(yī)學ppt腺病毒載體：雙鏈DNA，在基因轉移中的應用相當廣泛。其優(yōu)點是易制備、高滴度、宿主廣、能感染非增殖細胞，低毒，不整合入機體細胞染色體，容量達36kb，但免疫原性強，表達時間短。

33醫(yī)學ppt改進的腺病毒載體1.早期AVE1+E3區(qū)缺失的有限性2.E2,E4區(qū)的置換:Wilson報道:溫度敏感E2突變AV降低免疫性,表達延長;未根本解決；制備293/E2A,293/E4細胞;相應載體;擴大容量;降低毒性和免疫性;效果有限;滴度下降.293T細胞:轉染SV40大T抗原34醫(yī)學ppt降低腺病毒免疫原性

腺病毒載體插入B7反義基因,阻斷共刺激途徑;腺病毒載體插入ICAM-1反義基因,阻斷粘附因子作用.腺病毒載體插入CTLA4基因,胞內結合B7.

腺病毒載體插入MHC-I,II基因的調控基因反義片段.35醫(yī)學ppt增強腺病毒的感染效率腺病毒與葡聚糖,聚凝胺,多聚賴氨酸,脂質體混合感染細胞,組織,提高感染效率10-100倍,降低炎癥反應.重組腺病毒感染前,進行腺病毒封閉不同亞型腺病毒互換應用.36醫(yī)學pptaaaaaaEfficiency+++PersistenceSpecificityToxicity++RecombinantAdenovirusesApproachesGenerationIGenerationIIIHybridadenos:Adeno-RVAdeno-AAVAdeno-Transposase

Advantages/Limitations8KbcapacityGenerationI

>30KbcapacityGenerationIII

Adenocanbegrownatveryhightiters,

HoweverDonotintegrateCancontainRCAsAretoxic/immunogenicExamplesOTCdeficiency(clin,---)CysticFibrosis(clin,---)Oncolyticviruses(clin,+++)37醫(yī)學ppt腺病毒載體研究進展與展望進展展望Ad載體用于治療性的angiogenesis降低毒性的方法將繼續(xù)獲得進展,取得進展最終將提供安全的Ad載體系統(tǒng).Ad引起的毒性問題得到進一步認識靶向方法的應用將進一步改進.提高Ad載體的靶向性，改善療效缺陷型的Ad載體生產將不斷進展.新一代Ad載體能降低毒性，改善表達將面臨再次注射的挑戰(zhàn)38醫(yī)學ppt反轉錄病毒載體基因轉移系統(tǒng)輔助細胞:

將含有病毒結構基因但缺失了順式包裝信號的缺陷型反轉錄病毒導入哺乳動物細胞（ψ—2，PA317，ψCRIP）?？僧a生病毒包裝蛋白但不產生病毒顆粒；反轉錄病毒載體:以外源目的基因替換病毒結構基因,含病毒包裝信號輔助細胞提供載體包裝蛋白，使后者產生含外源目的基因的病毒顆粒39醫(yī)學ppt

缺陷型的反轉錄病毒顆粒的RNA進入靶細胞后，變成前病毒并整合到宿主細胞的染色體上，可穩(wěn)定表達外源基因。反轉錄病毒只能感染處于增殖期的細胞。40醫(yī)學pptaaaaaaPersistenceSpecificityEfficiencyToxicityApproachesMurineRetrovirusesVSV-pseudotypedRVLentivirusesSelf-inactivatingRVCombinationvirusesAdvantages/Limitations9Kbcapacity+integrationthroughtranspositionalsoinquiescentcells(HIV),permitinprinciplelong-termtreatments,howeverdisturbedby:InsertionalmutagenesisGenesilencingHighmutationrateLowtiterofproductionExamplesSCID(IL2Rdefect,Paris)(clin,+++)AdenosineDeaminasedeficiency(clin,+++!!!)Parkinson(preclin,+++)Anticancer(clin+/-)RecombinantRetroviruses(includesHIV-based)41醫(yī)學ppt

反轉錄病毒安全性問題病毒感染的可能性病毒在靶細胞基因組整合可導致:

破壞細胞正常生長必需的抑癌基因

LTR激活原癌基因染色體重排激活原癌基因42醫(yī)學ppt腺相關病毒（AAV）載體：4.7kb單鏈DNA，結構為ITR-rep-cap-ITR，病毒基因組簡單，易于消除，可降低細胞毒性和T-淋巴細胞反應的危險性；病毒DNA可在人19號染色體上進行位點特異性整合，需要輔助因子出現才復制；其Rep蛋白可能介導這種定向整合宿主范圍寬，易感染造血干細胞，能潛伏感染非分裂期細胞；在動物模型中表達可持續(xù)半年以上。但AAV載體接納的外源DNA小于4.5kb，載體整合效率較低，制備困難43醫(yī)學pptaaaaaaEfficiencyPersistenceSpecificityToxicityRecombinantadeno-associated-virus(AAV)ApproachesHelper-dependentproductionHelperindependentproductionCis-complementingvectorsCo-infectionAdvantages/LimitationsPersistenceinthegenomepermitslong-termexpression,hightitersareeasilyobtained,immunogenicityisverylow,HoweverSmallcapacity(<4.5kb)whichdoesnotallowtoaccommodatelargegenesorgeneclusters.ExamplesHemophiliaB(clin,animal,+++)Gaucher(clin,animal,+++)BrainIschemia(animal,+++)Cysticfibrosis(animal,+/-)44醫(yī)學pptLentivirus（慢病毒載體）優(yōu)點:感染非分裂細胞，有RV優(yōu)勢結構:LTR-gag-pol--env--LTR

TARtat,rev,tev應用:宿主廣泛;艾滋病基因治療問題:安全性45醫(yī)學pptHSV病毒雙鏈DNA,包括:HSV-1,2,VZV,EB,CMV.

主要以HSV-1型為主.HSV載體含包裝信號,復制位點等順序結構,HSV病毒啟動子,目的基因表達框架.由輔助病毒共轉染輔助細胞M64A,制備重組病毒.

輔助病毒的改造:IE3溫度敏感突變型;

缺失突變型:IE3缺失;

應用:感染心肌細胞,神經元,神經膠質細胞.VEGF---血管形成;腦腫瘤;帕金森病

46醫(yī)學pptaaaaaaRecap:currentlimitationsofpopularvectorsAdenovirus-nopersistence-limitedpackagingtoxicity,immunogenicityRetrovirus(incl.HIV)-limitedpackaging-randominsertion-unstablegenomeGeneral-antibodyresponse-limitedpackaging-genesilencingSolutions:-syntheticviruses(“Virosomes”)Biolisticbombardmentorlocaldirectinjection-limitedareaElectroporation-limitedorganaccessLiposomes,genecorrection&Co.-veryinefficienttransferGeneral-lowtransferefficiency-noorlittlegenomicintegrationSolutions:-improvedliposomeswithviralproperties(“Virosomes”)47醫(yī)學ppt非病毒載體除了基因轉移的效率比較低以外，比病毒載體具有更多的優(yōu)越性，將來會有viral-like,butartificialvectors.48醫(yī)學pptPharmacologicalconsiderationsforDNAtransfer

ClassicaldrugMW50-500DaltonsSyntheticallypreparedRapiddiffusion/actionOraldeliverypossibleCellulardelivery:

-actatcellsurface

-permeatecellmembrane

-importedthroughchannelsCanbedeliveredassolublemolecules?ngstrom/nmsizerapidlyreversibletreatment49醫(yī)學pptMw20,000-100,000DaBiologicallypreparedSlowerdiffusion/actionOraldeliverynotpossibleCellulardelivery

-actextracellularly

CanbedeliveredassolublemoleculesnmsizerapidlyreversibletreatmentProteindrug50醫(yī)學pptNucleicacidMwNx1,000,000DaBiologicallypreparedSlowdiffusionOraldeliveryinconceivableCellulardelivery:

-nomembranetranslocation

-nonucleartranslocation

-nobiologicalimportMustbedeliveredascomplexcarrierparticles50-200nmsizeslowlyornotreversible51醫(yī)學pptTherapywithnucleicacidsrequiresparticulatedformulationismuchmorecomplexthanpreviousdrugdeliverieshasadifferentdegreeofreversibility(dosageproblem)52醫(yī)學ppt2、非病毒及物理方法脂質體介導法磷酸鈣轉染法機械法（顯微注射、基因槍等）DEAE-葡聚糖和polybrene轉染法電穿孔法超聲波輔助法納米材料等53醫(yī)學ppt中性脂質體

由脂類形成的可高效包裝DNA的人造單層膜，其結構和性質與細胞膜極為相似，二者易于融合，細胞的內吞作用使其進入細胞，操作簡單，轉染效率可高達50%，可用于體內試驗，但目的基因表達不穩(wěn)定54醫(yī)學ppt陽離子脂質體：陽離子脂質體在水中可形成大小約100-400nm單層脂質體。其帶正電，帶負電的DNA可自動結合到帶正電的脂質體上，形成DNA-陽離子脂質體復合物，與細胞膜作用使DNA進入細胞。在該系統(tǒng)體內基因導入效率低，且無靶向性。在體內應用，除腫瘤瘤內注射外，其前景仍存在問題。55醫(yī)學ppt56醫(yī)學ppt磷酸鈣轉染法：將氯化鈣、目的DNA和磷酸緩沖液混合，沉淀形成含有DNA的極小的不溶性磷酸鈣顆粒。磷酸鈣-DNA復合物粘附到細胞膜，通過胞飲進入受體細胞的細胞質，轉染過程簡單有效，適用于貼壁、非貼壁細胞，轉染效率可達20%左右，但目的基因表達不穩(wěn)定57醫(yī)學ppt機械法

如顯微注射和基因槍（biolisticparticle）。顯微注射使用一根細針頭將外源DNA直接轉入受體細胞核。基因槍使用高壓DNA分子導入細胞。

基因槍工作原理顯微注射58醫(yī)學pptaaaaaaSpecificityEfficiencyPersistenceToxicityApproachesAntisenseRibozymes/DNAzymesTriplehelixDecoy/competitorsGene-correctingoligosAdvantages/Limitationstheseproceduresmaybesuitablefor:handlingdominantdefectstransienttreatments(genemodulation)permanenttreatments(genecorrection)ExamplesAnticancer(clin,preclin.,+/-)Restenosis(clin,+++)MuscularDistrophy(animal,+++)Oligonucleotides59醫(yī)學ppt電穿孔法通過將細胞暴露在短暫的高場強電脈沖中轉導目的DNA。細胞懸浮液置于電場中會誘導沿細胞膜的電壓差異，這種電壓差異會導致細胞膜暫時穿孔。一般，成功的電穿孔過程都伴隨高水平（50%或更高）的毒性。60醫(yī)學pptDEAE-葡聚糖和polybrene轉染法

帶正電的DEAE-葡聚糖或polybrene多聚體復合物和帶負電的外源DNA分子作用，使得DNA可以結合在細胞表面。通過使用DMSO或甘油獲得的滲透休克將外源DNA導入。兩種試劑都已成功用于轉染。DEAE-葡聚糖僅限于瞬時轉染。61醫(yī)學pptaaaaaaSpecificityPersistenceEfficiencyToxicityApproachesNakedDNAinjection/biolisticNakedDNA+pressureNakedDNA+electroporationLiposomalformulationsCombinationsAdvantages/LimitationsUnlimitedsizecapacity+lowerimmunogenicityandlowerbio-riskofnonviralformulationsisdisturbedbyLowefficiencyofgenetransferEvenlowerstableintegrationExamplesCriticallimbIschemia(clin,+++)CardiacIschemia(clin,+/-)Vaccination(clin,+/-)Antirestenosis(preclin.+/-)Naked/complexedDNA62醫(yī)學pptaaaaaaIdealpropertiesofasystemicallydelivered

non-viralformulationStabilityparticleshouldresistseruminactivationparticleshouldbeinerttoimmuneinactivation

Addressabilityparticleshouldpossessavascularaddressingsignatureparticleshouldbearatissue-dockingspecificityDNAconstructshouldincludetissue-specificregulatoryelements

Efficiencycargoshouldbeprotectedfromcytoplasmicinactivation(ex.lysosomes)cargoshouldcontainnuclear-translocatingsignalsDNAcargoshouldincludegenome-integrationfunctionsDNAelementmustbeguaranteedtofunctionaftergenomicintegration(nosilencing)63醫(yī)學pptOtherpropertiesParticleshouldnotincludeimmunogenic/toxicsurfacescargoshouldnotencodeimmunogenic/toxicproductsCargoshouldincludeanti-apoptoticfunctions∴

severalindependentproblemsmustbesolvedforanonviralformulationtobesuitableforclinicaltreatmentandforindustrialproductionmostviralvectorsincludemany,ifnotallthoseproperties64醫(yī)學ppt

裸DNA基因轉移和治療進展 展望裸DNA肌肉注射，活體轉移 非病毒基因轉移越來越重要電擊促進質粒DNA進入肌肉皮膚 未來幾年遺傳病臨床試驗將首選血友病血管內導入質粒DNA，肝細胞基因轉移 Duchenne肌營養(yǎng)不良，關節(jié)炎等尾靜脈導入pDNA，簡單有效，轉染大、嚙齒類的尾靜脈注射作為快速檢測表小鼠肝細胞達狀況和基因治療方法廣泛應用pDNA表達載體可高水平持續(xù)表達 血管內裸DNA導入為細胞有效涉入pDNA RNAi將在基因治療領域中成為重要手裸DNA導入法已經在臨床研究（PAOD，段外周動脈閉鎖癥）SiRNA可通過血管內基因轉移法有效導入Knockout小鼠65醫(yī)學pptaaaaaa∴problemsthatmustbesolvedtobesuitableforclinicaltreatmentandforindustrialproductionaredifferentbetweenviralandnon-viralvectorswhenignoringtheirlowefficiency,nonviralvectorsappearslargelysuperiorMostrelevantissuesinthetwomain'vectorology'sectors(viralversusnonviral)ViralvectorsPackagingcapacityfrom4to30kbproblemforsomelargegenes(ex.dystrophingeneorCFTRgene)importanttoxicload:ratioinfectious/non-infectiousparticlesfrom1/10to1/100strongimmunogenicity:capsidandenvelopeproteins,residualviralgenescontaminants:replication-competentviruses(ex.wildtyperevertantviruses)Viralamount(titre)obtainablewithrecombinants(ex.10exp5=poor,10exp10=excellent)Complexityofproduction(existenceornotofpackagingcellsystems)Emotionalproblemslinkedtopathogenicityofdonorvectors(ex.lentiviruses)NonviralvectorsPackagingcapacitynotanissue,evenverylargeconstructscanbeused(exampleentirelociupto150kb)minortoxicload:smallpercentageofnonrelevantadventitiousmaterialsmoderateimmunogenicity:methylationstatusofDNA(exampleCpGmotifs)contaminants:adventitiouspathogensfrompoorDNApurification(exendotoxins)AmountofDNAmoleculesisusuallynotaproblem,theothercomponentsdependsonchemicalsynthesisNoparticularcomplexity,exceptforspeciallyformulatedliposomesnoparticularemotionalproblemslinkedtothenatureofthereagents66醫(yī)學pptaaaaaaRandomintegratingvectorsr-lentivirusesr-retrovirusesr-AAVplasmids(lowfrequency)plasmids+transposase(eg'sleepingbeauty')

Transient,nonintegratingvectorsadenovirusplasmidRNAvirusbasedoligonucleotides(SiRNA,antisense,ribozymes)artificialchromosomes

Genecorrectionvectorschimeroplasts(RNA-DNAchimericoligos)singlestrandedDNA(homologousrecom)genotoxicnon-genotoxicSpecificallyintegratingvectorshybri