1、慢病毒载体的构建慢病毒载体的构建Gateway to lentiviral vectorIntroduction 慢病毒（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 （Human immunodeficiency virus） 、EIAV （Equine infectious anemia virus） 、 FIV （Feline immunodeficiency virus） 、 SIV （Simian immunodefic
2、iency virus） 。其中研究最多最为透彻的是HIV。 Lentiviruses life cycle 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
3、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 。Mitrophanous K, Gene Ther 5(11):14811487(1999)Lentiviral Vector SystemsUnder Development Primate Human immunodeficiency virus (HIV) Simian immunodeficiency virus (SIV) Non-primate Feline immunodeficiency virus (FIV) Equine infec
4、tious anemia virus (EIAV)The outline of this ppt 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：一一. HIV-1 life cycle. HIV-1 life cycle 二二. HIV-1. HIV-1基因结构和病毒颗粒结构基因结构和病毒颗粒结构三三. .载体系统构建的基本原理载体系统构建的基本原理 四四. .载体系统的设计载体系统的设计五五. .包装系统的设计包装系统的设计 - HIV-1-Derived lentiviral vector production ,- HIV-1-Derived lentiviral v
5、ector production , concentration & titration concentration & titration 1. HIV-1-Derived lentiviral vector production & titration 1. HIV-1-Derived lentiviral vector production & titration 2.HIV-1 Vector Production-Supernatant recoveries & concentrat 2.HIV-1 Vector Production-Supernatant recoveries &
6、concentrat六六.The difference of Packaging Cells for LVV with other retroviruses.The difference of Packaging Cells for LVV with other retroviruses七七.The Development of LV Vectors.The Development of LV Vectors八八.Advantages and Disadvantages .Advantages and Disadvantages of Lentiviralof Lentiviral vecto
7、rs vectors九九.The comparison of Lentiviral.The comparison of Lentiviral vectors with other vectors vectors with other vectors一一 HIV-1 life cycle 二二 HIV-1HIV-1基因结构基因结构 gag, -群抗原基因，编码核心蛋白p24 pol -多聚酶基因，编码多聚酶； env-包膜蛋白基因，编码包膜蛋白gp120 及gp41； tat - 基因反式激活因子对HIV-1基因其正调控作用 rev,- 病毒蛋白表达调节因子，能增加gag和env基因对结构蛋白的
8、表达 vif,- 病毒感染因子, 其作用是在一些细胞因子的协下促进HIV-1在细胞内复 Vpr,- R蛋白能使HIV在巨噬细胞中增殖 vpu,- U蛋白，能促进HIV从细胞膜上释放 nef,-负因子，具有抑制HIV-1增殖作用二二 HIV-1HIV-1病毒颗粒结构病毒颗粒结构三三 载体系统构建的基本原理载体系统构建的基本原理 HIV-1基因组中如包装信号、长末端重复序列的顺式作用元件与编码反式作用蛋白的序列进行分离。即从病毒基因组中将反式gag, pol, and env基因(其他辅助基因省去)从病毒中分离出而用我们感兴趣的外源性目的基因代替，剩下的顺式作用元件在病毒复制周期中-逆转录、整合、
9、转录和包被-可以被其它病毒或细胞蛋白识别。 Fig.1 慢病毒载体的顺式作用元件四四 载体系统的设计载体系统的设计慢病毒载体系统由三种不同的组成部分：包装结构、转移载体成分和包膜蛋白成分（Env表达结构）。包装部分由去除了包装、逆转录和整合所需的顺式作用元件的HIV-1基因组而构建,能反式提供产生病毒颗粒所需的蛋白;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)
10、 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（SV 40）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po l、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) Components of the lentiviral systemExpression vectorPackagingvectorEnvelop/Host Range vector五五 包装系统的设计包装系统的设计 - H
11、IV-1-Derived lentiviral vector- HIV-1-Derived lentiviral vector production ,concentration & titration production ,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
12、毒：VECTOR CONSTRUCTPACKAGING CONSTRUCTENVELOPE CONSTRUCT1. HIV-1-Derived lentiviral vector production & titrationPackaging ConstructTransfer Vector ConstructEnvelope ConstructTri-Transfection(CaPO4)293T cellsPseudoparticles Recovery(filtration 0.45m)Supernatants at 48h and 72h p.t.Concentration by Ul
13、tracentrifugationVector Titer DeterminationTransduced CellsVirionsFACS(TU/ml)qPCR SybRGreen(proviral DNA/cell)p24 ELISA(ng p24/ml)All plasmids containa SV40 replication origin- Episomal statusIPPP2.HIV-1 Vector ProductionSupernatant recoveries & concentration 293T producer cellsD0Tri-transfectionD1M
14、edium exchangeD2 & D3SupernatantharvestsFiltration 0.45mConcentration byultracentrifugationD3 supernatantsSW28Pool 212AliquotsD2 supernatantsSW28Pool 112Store at 4CAliquots3SW55Pool 1 & Pool 2Pool 3AliquotsIn the evening 六六 The difference of Packaging Cells for LVV The difference of Packaging Cells
15、for LVV with other retroviruseswith other retroviruses 七 The Development of LV VectorsThe 1st GenerationCMV (cytomegalovirus) immediate-early enhancer/promoterVSV-G (Vesicular stomatitis virus glycoprotein envelope)A cellular polyA was used to replace the 3 LTRvpu from HIV was deletedThe Development
16、 of LV VectorsThe 2nd GenerationBiosafety issue:Additional accessory genes were deleted (vif, vpu, vpr and nef)The generation of RCL (replication competent lentiviruses) was reducedThe Development of LV VectorsThe 3rd GenerationSelf-inactivating (SIN) vectorsFurther improvement in biosafetyTat was d
17、eletedRev was put into a separate plasmidU3 of the 5 LTR was replaced by the CMV IE promoter/enhancerThe possibility of generating RCL is further reduced八八 Advantages and Disadvantages of Lentiviral vectors 1.Potential for long term therapeutic expression: incorporates into the genome of target cell
18、s 2.High efficiency gene transduction 3.Transduce several nondividing cells 4.Broad application in vivo / ex vivo1.Potential for insertional mutagenesis 2.Unable to transduce all nondividing cells (e.g. Hepatocytes) 3.Safety issues 1).HIV-based vectors must overcome a great social barrier 2).Limited
19、 knowledge of other lentivirus poses a risk for recombinationAdvantages and Disadvantages of Lentiviral vectors LVs are considered attractive CNS gene transfer tools due their capacity to transduce slowly or nondividing cells in the brain 23, the extremely low probability of occurrence of replicatio
20、n-competent retroviruses 46, the lack of expression of viral genes 1, the relatively large cloning capacity 1, the ability to expand the host range by pseudotyping LVs with a variety of envelopes 7,8, and the possibility of incorporating complex expression cassettes 9. LVs have been demonstrated to
21、transduce most cell-types within the CNS in vivo, including neurons, astrocytes, adult neuronal stem cells, oligodendrocytes, and glioma cells 2,1012. 九The comparison of Lentiviral vectors with other vectorsReferences 1. Naldini, L., et al. (1996). In vivo gene delivery and stable transduction of no
22、ndividing cells by a lentiviral vector. Science 272: 263 267. 2. Blomer, U., Naldini, L., Kafri, T., Trono, D., Verma, I. M., and Gage, F. H. (1997). Highly efficient and sustained gene transfer in adult neurons with a lentivirus vector. J. Virol. 71: 6641 6649. 3. Naldini, L., Blomer, U., Gage, F.
23、H., Trono, D., and Verma, I. M. (1996). Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93: 11382 11388. 4. Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997).
24、 Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15: 871 875. 5. Zufferey, R., et al. (1998). Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72: 9873 9880. 6. Dull, T., et al. (1998). A third-generation len
25、tivirus vector with a conditional packaging system. J. Virol. 72: 8463 8471. 7. Watson, D. J., Kobinger, G. P., Passini, M. A., Wilson, J. M., and Wolfe, J. H. (2002). Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope
26、proteins. Mol. Ther. 5: 528 537. 8. Wong, L. F., et al. (2004). Transduction patterns of pseudotyped lentiviral vectors in the nervous system. Mol. Ther. 9: 101 111. 9. Wiznerowicz, M., and Trono, D. (2005). Harnessing HIV for therapy, basic research and biotechnology. Trends Biotechnol. 23: 42 47.
27、10. Consiglio, A., et al. (2004). Robust in vivo gene transfer into adult mammalian neural stem cells by lentiviral vectors. Proc. Natl. Acad. Sci. USA 101: 14835 14840. 11. Jakobsson, J., Ericson, C., Jansson, M., Bjork, E., and Lundberg, C. (2003). Targeted transgene expression in rat brain using lentiviral vectors. J. Neurosci. Res. 73: 876 885. 12. Miletic, H., et al. (2004). Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycoproteins. Hum. Gene Ther. 15: 1091 1100.References HULUN BEIER PRAIRIE
