《慢病毒简介》PPT课件.ppt

1,慢病毒,2,慢病毒载体概况HIV-1的基因结构慢病毒载体历史与构建重组慢病毒的产生慢病毒在中枢神经系统中的应用慢病毒在造血干细胞系统中的应用慢病毒在眼科疾病治疗中的应用,3,1.慢病毒载体概况,慢病毒是逆转录病毒科亚科之一，分为灵长类和非灵长类慢病毒。灵长类慢病毒包括HIV-1，HIV-2，猴免疫缺陷病毒（SIV），非灵长类慢病毒包括猫免疫缺陷病毒（FIV），牛免疫缺陷病毒（BIV），马免疫缺陷病毒（EIAV）等，其中HIV研究最为透彻。研究表明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。这一特性是慢病毒成为基因治疗的转移载体。,4,2.HIV-1的基因结构,HIV-1为双链RNA病毒，共有9个基因。gag基因编码病毒的核心蛋白，包括基质蛋白、衣壳蛋白、核衣壳蛋白。pol基因编码病毒复制所需的酶，如反转录酶、整合酶、蛋白酶。env基因编码病毒的包膜糖蛋白，决定病毒感染宿主的靶向性。rev编码的蛋白调节gag、pol、env的表达水平。tat编码的蛋白参与RNA转录的控制。4个辅助基因vif、vpr、vpu、nef编码的蛋白则作为毒力因子参与宿主细胞的识别和感染。两端为长末端重复序列(LTR)，内含复制所需的顺式作用元件。,编码病毒的基本结构,调节基因,5,3.慢病毒载体历史与构建,3.1第一代HIV-1来源的慢病毒载体 以Naldini及Kafri构建的三质粒系统为代表，该系统由包装质粒、包膜质粒及载体质粒3种质粒组成。包装质粒是HIV-1前病毒基因组5端LTR由巨细胞病毒早期启动子取代，3LTR由SV40 polyA序列取代。包装成分分别构建在两个质粒上，一个表达gag和pol，另一个表达env。载体质粒携带了5端LTR，和全部5端非翻译区域，另外还带有rev应答元件（RRE）。包膜表达质粒使用水疱性口炎病毒糖蛋白G基因(VSV-G)用来代替了原病毒的env基因。,6,3.2第二代HIV-1来源的慢病毒载体 1997年,Zufferey等将包装质粒上的vif、vpr、vpu和nef基因(即辅助基因)敲除,从而得到的,其他方面与第一代载体系统一致。,7,3.3第三代HIV-1来源的慢病毒载体 为减少复制型病毒的产生，可通过减少辅助质粒与载体质粒的同源性，或者将gag/pol和rev编码序列隔离,分散在不同的质粒上。这样的包装系统由四质粒代替原有的三质粒包装系统。质粒一携带了gag/pol编码序列及RRE;质粒二包含了编码rev的序列;质粒三是载体质粒;质粒四表达env。,8,3.4自身失活型（SIN）慢病毒载体 SIN载体的构建是在原病毒载体基础上删除了病毒3端LTR的U3区增强子和启动子序列的片段。该区域出现突变则在HIV-1载体转录后，其5LTR会因为缺失HIV-1所需要的启动子和增强子序列而无法复制出完整长度的病毒基因组。,9,4.重组慢病毒的产生,常用瞬时转染法，即将包膜质粒，包装质粒和载体质粒共转染人胚肾293T细胞直接产生生产细胞。最后慢病毒分泌到培养基中进行培养而得到大量载体慢病毒。,10,慢病毒在中枢神经系统中的应用,11,1.Introduction,Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology,as well as pathogenesis and treatments for various neurodegenerative diseases.Lentiviral vectors are valuable tools for neurobiology research owing to their ability to transduce nondividing cells,such as neurons,and to introduce therapeutic or reporter genes into central nervous system(CNS)cells in vivo and in vitro.重组病毒载体已被用于研究各种发育神经生物学中的基础问题，以及各种神经退行性疾病的发病机理和治疗。慢病毒载体能转导分裂的细胞，如神经元，并介导中枢神经系统（CNS）细胞在体内和体外基因治疗或报道基因而作为神经生物学研究的有价值的工具。,12,1.1.Lentiviral Gene Delivery to CNS Cells,Lentivirus preintegration complexes interact with the nuclear pore and undergo active transport into the nucleus of nondividing cells,where the proviral DNA is integrated into the genomic DNA of the host cell.This feature is the primary reason why lentiviruses are being devel-oped as gene-transfer vectors for postmitotic cells in the CNS.慢病毒整合前复合物与核孔相互作用进入细胞核分裂的细胞，其中的前病毒DNA整合到宿主细胞的基因组DNA并进行主动运输。此功能是为什么慢病毒在有丝分裂后的细胞在中枢神经系统的基因转移载体的主要原因。,13,Self-inactivating(SIN)vectors reduce the probability of oncogenesis by pro-moter insertion.In SIN vectors,viral promoter activity is deleted from the inte-grated provirus by deletions in the U3 region of the 3 long terminal repeat(LTR)that are copied during reverse transcription to the 5LTR.自身失活型（SIN）慢病毒载体3端LTR的U3区启动子发生失活突变后，在逆转录过程中转移至5LTR。这样的载体整合入靶细胞，将不会产生完整长度的载体RNA，因此命名为“自身失活型载体”。,14,To increase gene delivery in brain,newer generations of lentiviral vectors incorporate the central poly-purine tract(cPPT),an approx 180 bp region derived from the gag region,which increases nuclear import of the proviral DNA and transduction efficiency in the brain.为了增加基因在脑中的传递，新一代的慢病毒载体包含cPPT，一个来自gag区约180bp的区域，从而增加了原病毒DNA进入核，也提高了在大脑中的转导效率。,15,1.2.Targeted Gene Delivery in the CNS Using Pseudotyped Lentiviral Vectors,Another feature of the lentiviral vector system is that the virions can carry a surface protein that bypasses the usual HIV receptors and co-receptors,thus changing or expanding the range of cell types that the vector can bind to and enter.This is done by pseudotyping,which involves replacing the HIV-1 envelope glycoprotein with an envelope glycoprotein from another virus,such as the vesicular stomatitis virus glycoprotein(VSV-G).慢病毒载体系统的另一个特征是携带病毒微粒的表面蛋白，包含HIV受体和共受体，从而改变或扩大的细胞类型的范围，使得该载体可以结合并且进入。这个模型涉及取代的HIV-1包膜糖蛋白与另一种病毒的包膜糖蛋白，如疱疹性口腔炎病毒糖蛋白（VSV-G）。,16,1.3.Lentiviral Vector Production,The development of stable packaging cell lines that produce high titers of lentiviral vectors has been hindered by the toxicity of constitutive VSV-G expression.For this reason,many groups continue to use transient triple transfection to generate their vector stocks.产生高滴度的慢病毒载体的稳定的包装细胞系的发展受到VSV-G表达的毒性的阻碍。因此，大多数人使用三质粒系统。,17,Three plasmids are required:encoding the envelope glycoprotein(most commonly VSV-G),encoding the packaging proteins(minimally including gag and pol,often including tat and rev on the same plasmid,and in some cases the accessory genes vif,vpr,vpu,and nef),encoding the genome(including the intact or self-inactivating LTRs,the packaging signal,the promoter and cDNA of interest and,in some cases,posttranslational regulatory elements and the central poly-purine tract(cPPT),which increase titer and expression.,18,2.Materials2.1.Transfection,1.A highly transfectable cell line such as human embryonic kidney 293T.2.Growth media for 293T cells.3.Poly-D-lysine.4.Borate buffer.5.Reagents for calcium phosphate transfection.,19,6.High-quality plasmid DNA:transfer plasmid,packaging plasmid,envelope plasmid purified through cesium chloride/ethidium bromide equilibrium centrifugation or an anion-exchange matrix.7.Polybrene,800 g/mL stock solution in PBS.8.Large polyallomer centrifuge tubes for concentrating the vector in a Beckman SW28 ul-tracentrifuge rotor.,20,2.2.Stereotactic Surgery,Anaesthesia mice.Surgical Preparation.Drilling and injection.Postoperative care.Perfusion.A manual for stereotaxic surgery such as Stereotaxic Surgery in the Rat.A mouse brain atlas such as The Mouse Brain in Stereotaxic Coordinates.,21,3.Methods,Transfection转染Collection and Concentration of the Viral Supernatant 收集和集中的病毒上清Titering Lentiviral Vectors 滴定慢病毒载体,22,慢病毒在造血干细胞系统中的应用,23,造血干细胞（HSC）具有自我更新和分化为血液及免疫系统中各种成熟细胞的能力，许多HSC疾病如遗传性、代谢性和感染性疾病或恶性肿瘤等有望通过基因治疗的方法得到纠正。目的基因转移HSC后，可随着HSC的自我更新和分化在体内长期表达，因此HSC是较理想的基因转移靶细胞。,24,研究表明，VSV-G假构型HIV-I载体可不经过对HSC的预刺激就能有效地将基因转移到人早期干细胞并能在重度联合免疫缺陷(SCID)鼠骨髓中稳定地长期表达，Miyoshi等构建VSV-G假构型HIV-I载体，以绿色荧光蛋白（GFP）作为标记基因，将新鲜分离的人脐血CD34+细胞在无血清及细胞因子培养基中转染5小时，然后植入经亚致死量照射的非肥胖型糖尿病/重度联合免疫缺陷(NOD/SCID)鼠，可在受鼠脾脏、骨髓及外周血GFP的长期表达。,25,Methods,Isolation of Human CD34+Hematopoietic Progenitor Cells 人CD34+造血祖细胞的分离Preparation of Lentiviral Vectors 慢病毒载体的制备Transduction of CD34+Cells by Lentiviral Vectors 慢病毒载体转导CD34+细胞Analysis of Transduced Human CD34+Cells in NOD/SCID Mice分析转人CD34+细胞的NOD/SCID小鼠,26,慢病毒在眼科疾病治疗中的应用,27,1.Introduction,The primary aim of gene transfer into the retinal cells has been to investigate the developmental mechanisms of the retinal cells or to reverse retinal diseases.Currently,lentivirus and adenoassociated virus vectors are being used for studying and correcting gene therapy of retinal degenerative diseases.视网膜细胞基因转导的主要目的是研究视网膜细胞的发病机制或逆转视网膜疾病。目前，慢病毒和腺病毒载体在被用于研究和纠正的视网膜变性性疾病的基因治疗。,28,Using an HIV vector carrying the green fluorescent protein(GFP)gene expressed from the cytomegalovirus(CMV)promoter,we showed that efficient and long-lasting gene expression could be obtained in the retina(Fig.1).Moreover,gene expression was restricted to the photoreceptor cells and was more efficient with the rhodopsin promoter.HIV载体携带绿色荧光蛋白（GFP）基因其通过巨细胞病毒（CMV）启动子的表达，可以在视网膜上得到高效和持久的基因的表达（图1）。此外，基因表达被限制在感光体细胞中，是更有效的视紫红质子。,29,Fig.1.Subretinal injection.视网膜下注射 scleral approach.巩膜方法(B)vitreous approach.玻璃体方法,30,2.Surgical Instruments,Injection for the Small EyeVitrectomy for the Large Eye 大眼睛的玻璃体手术治疗,31,Methods,Gene Delivery to the Subretinal Space of Rats and Mice大鼠和小鼠视网膜下腔的基因传递Gene Delivery to the Retina of Monkey 猴视网膜基因传递Gene Delivery to the Vitreous Space 基因传递至玻璃体空间Transfection Efficiency 转染效率,32,谢谢大家！,