慢病毒载体的构建方法课件.ppt

慢病毒载体的构建Gatewaytolentiviralvector,-献给勇敢的斗士,January2007,Introduction,慢病毒（Lentiviruses）属于逆转录病毒科。慢病毒核蛋白质前整合复合物具有噬核特性，病毒基因组运输至细胞核，从而使慢病毒可以感染和在非有丝分裂细胞中复制。这一特性使慢病毒成为基因治疗的转移载体。HIV（Humanimmunodeficiencyvirus）、EIAV（Equineinfectiousanemiavirus）、FIV（Felineimmunodeficiencyvirus）、SIV（Simianimmunodeficiencyvirus）。其中研究最多最为透彻的是HIV。,Lentiviruseslifecycle,慢病毒通过病毒衣壳糖蛋白与细胞膜上的特异受体结合而进入易感靶细胞。一旦与细胞膜上的特异受体结合，慢病毒膜和细胞膜融合后病毒核心释放入细胞浆里。病毒RNA逆转录合成双链线性DNA而转运之细胞核。线性病毒DNA永久性的整合入染色体DNA（宿主基因组）中，形成前病毒，成为永久的遗传成分，在细胞周期中与靶细胞基因一样进行复制，转给子代细胞。前病毒DNA转录成RNA后再转运至胞浆，在胞浆里RNA翻译成病毒蛋白。病毒前结构蛋白和复制酶与病毒RNA组装成新的病毒核心，从包装细胞获得病毒包膜蛋白后以出芽的方式从细胞膜上释放出。病毒前结构蛋白经进一步处理而最终形成成熟的具有感染性的子代病毒颗粒。这些特性是慢病毒成为基因治疗转运工具的重要原因。,MitrophanousK,GeneTher5(11):14811487(1999),LentiviralVectorSystemsUnderDevelopment,PrimateHumanimmunodeficiencyvirus(HIV)Simianimmunodeficiencyvirus(SIV)Non-primateFelineimmunodeficiencyvirus(FIV)Equineinfectiousanemiavirus(EIAV),Theoutlineofthisppt,接下来将以HIV为例从以下方面说明慢病毒载体构建方面的基础知识：一.HIV-1lifecycle二.HIV-1基因结构和病毒颗粒结构三.载体系统构建的基本原理四.载体系统的设计五.包装系统的设计-HIV-1-Derivedlentiviralvectorproduction,concentration载体部分与包装成分互补,仅含有包装、逆转录和整合所需的HIV-1顺式作用元件,同时具有异源启动子控制下的MCS及在此位点插入的目的基因。三种表达结构均以细菌质粒的形式保存，能转染到哺乳动物细胞内产生复制缺陷性病毒原种。为降低包装成分和载体成分同源重组产生有复制能力的慢病毒(RCL)的可能性,将包装成分的5LTR换成巨细胞病毒(CMV)立即早期启动子,3LTR换成猿猴空泡病毒40（SV40）polyA位点等，而将包装成分分别构建在两个质粒上,即一个表达gag和pol、另一个表达env。以HIV-1为基础的慢病毒载体而言，病毒颗粒的核心和酶成分来自于HIV-1,而包膜蛋白来自于异源性病毒,大多是水泡性口炎病毒(VSV-G),Componentsofthelentiviralsystem,Expressionvector,Packagingvector,Envelop/HostRangevector,五包装系统的设计-HIV-1-Derivedlentiviralvectorproduction,concentration&titration,重组慢病毒的产生：瞬时转染法，即将包装结构和载体结构瞬时共转染法如293T高表达细胞系而产生重组慢病毒。此法非常成功，大多实验室采用此法。包膜质粒、包装质粒与载体质粒共转(多用磷酸钙共沉淀法)染293T细胞直接产生生产细胞。最后重组慢病毒分泌到培养基中进行培养而得到大量载体慢病毒。例如用以下三质粒来包装产生重组慢病毒：,VECTORCONSTRUCT,PACKAGINGCONSTRUCT,ENVELOPECONSTRUCT,2019/12/19,13,可编辑,1.HIV-1-Derivedlentiviralvectorproduction&titration,2.HIV-1VectorProductionSupernatantrecoveries&concentration,Intheevening,六ThedifferenceofPackagingCellsforLVVwithotherretroviruses,七TheDevelopmentofLVVectorsThe1stGeneration,CMV(cytomegalovirus)immediate-earlyenhancer/promoterVSV-G(Vesicularstomatitisvirusglycoproteinenvelope)AcellularpolyAwasusedtoreplacethe3LTRvpufromHIVwasdeleted,TheDevelopmentofLVVectorsThe2ndGeneration,Biosafetyissue:Additionalaccessorygenesweredeleted(vif,vpu,vprandnef)ThegenerationofRCL(replicationcompetentlentiviruses)wasreduced,TheDevelopmentofLVVectorsThe3rdGeneration,Self-inactivating(SIN)vectorsFurtherimprovementinbiosafetyTatwasdeletedRevwasputintoaseparateplasmidU3ofthe5LTRwasreplacedbytheCMVIEpromoter/enhancerThepossibilityofgeneratingRCLisfurtherreduced,八AdvantagesandDisadvantagesofLentiviralvectors,1.Potentialforlongtermtherapeuticexpression:incorporatesintothegenomeoftargetcells2.Highefficiencygenetransduction3.Transduceseveralnondividingcells4.Broadapplicationinvivo/exvivo,1.Potentialforinsertionalmutagenesis2.Unabletotransduceallnondividingcells(e.g.Hepatocytes)3.Safetyissues1).HIV-basedvectorsmustovercomeagreatsocialbarrier2).Limitedknowledgeofotherlentivirusposesariskforrecombination,AdvantagesandDisadvantagesofLentiviralvectors,LVsareconsideredattractiveCNSgenetransfertoolsduetheircapacitytotransduceslowlyornondividingcellsinthebrain23,theextremelylowprobabilityofoccurrenceofreplication-competentretroviruses46,thelackofexpressionofviralgenes1,therelativelylargecloningcapacity1,theabilitytoexpandthehostrangebypseudotypingLVswithavarietyofenvelopes7,8,andthepossibilityofincorporatingcomplexexpressioncassettes9.LVshavebeendemonstratedtotransducemostcell-typeswithintheCNSinvivo,includingneurons,astrocytes,adultneuronalstemcells,oligodendrocytes,andgliomacells2,1012.,九ThecomparisonofLentiviralvectorswithothervectors,References,1.Naldini,L.,etal.(1996).Invivogenedeliveryandstabletransductionofnondividingcellsbyalentiviralvector.Science272:263267.2.Blomer,U.,Naldini,L.,Kafri,T.,Trono,D.,Verma,I.M.,andGage,F.H.(1997).Highlyefficientandsustainedgenetransferinadultneuronswithalentivirusvector.J.Virol.71:66416649.3.Naldini,L.,Blomer,U.,Gage,F.H.,Trono,D.,andVerma,I.M.(1996).Efficienttransfer,integration,andsustainedlong-termexpressionofthetransgeneinadultratbrainsinjectedwithalentiviralvector.Proc.Natl.Acad.Sci.USA93:1138211388.4.Zufferey,R.,Nagy,D.,Mandel,R.J.,Naldini,L.,andTrono,D.(1997).Multiplyattenuatedlentiviralvectorachievesefficientgenedeliveryinvivo.Nat.Biotechnol.15:871875.5.Zufferey,R.,etal.(1998).Self-inactivatinglentivirusvectorforsafeandefficientinvivogenedelivery.J.Virol.72:98739880.6.Dull,T.,etal.(1998).Athird-generationlentivirusvectorwithaconditionalpackagingsystem.J.Virol.72:84638471.7.Watson,D.J.,Kobinger,G.P.,Passini,M.A.,Wilson,J.M.,andWolfe,J.H.(2002).TargetedtransductionpatternsinthemousebrainbylentivirusvectorspseudotypedwithVSV,Ebola,Mokola,LCMV,orMuLVenvelopeproteins.Mol.Ther.5:528537.,8.Wong,L.F.,etal.(2004).Transductionpatternsofpseudotypedlentiviralvectorsinthenervoussystem.Mol.Ther.9:101111.9.Wiznerowicz,M.,andTrono,D.(2005).HarnessingHIVfortherapy,basicresearchandbiotechnology.TrendsBiotechnol.23:4247.10.Consiglio,A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