慢病毒簡介

1、1,慢病毒載體概況HIV-1的基因結(jié)構(gòu)慢病毒載體歷史與構(gòu)建重組慢病毒的產(chǎn)生慢病毒在中樞神經(jīng)系統(tǒng)中的應(yīng)用慢病毒在造血干細胞系統(tǒng)中的應(yīng)用慢病毒在眼科疾病治療中的應(yīng)用,2,1.慢病毒載體概況,慢病毒是逆轉(zhuǎn)錄病毒科亞科之一，分為靈長類和非靈長類慢病毒。靈長類慢病毒包括HIV-1，HIV-2，猴免疫缺陷病毒（SIV），非靈長類慢病毒包括貓免疫缺陷病毒（FIV），牛免疫缺陷病毒（BIV），馬免疫缺陷病毒（EIAV）等，其中HIV研究最為

2、透徹。研究表明慢病毒核蛋白質(zhì)前整合復(fù)合物具有噬核特性，病毒基因組運輸至細胞核，從而使慢病毒可以感染和在非有絲分裂細胞中復(fù)制。這一特性是慢病毒成為基因治療的轉(zhuǎn)移載體。,3,2.HIV-1的基因結(jié)構(gòu),HIV-1為雙鏈RNA病毒，共有9個基因。 gag基因編碼病毒的核心蛋白，包括基質(zhì)蛋白、衣殼蛋白、核衣殼蛋白。pol基因編碼病毒復(fù)制所需的酶，如反轉(zhuǎn)錄酶、整合酶、蛋白酶。 env基因編碼病毒的包膜糖蛋白，決定病毒感染宿主的靶向性。re

3、v編碼的蛋白調(diào)節(jié)gag、pol、env的表達水平。tat編碼的蛋白參與RNA轉(zhuǎn)錄的控制。4個輔助基因vif、vpr、vpu、nef編碼的蛋白則作為毒力因子參與宿主細胞的識別和感染。兩端為長末端重復(fù)序列(LTR)，內(nèi)含復(fù)制所需的順式作用元件。,編碼病毒的基本結(jié)構(gòu),調(diào)節(jié)基因,4,3.慢病毒載體歷史與構(gòu)建,3.1第一代HIV-1來源的慢病毒載體 以Naldini及Kafri構(gòu)建的三質(zhì)粒系統(tǒng)為代表，該系統(tǒng)由包裝質(zhì)粒、包膜質(zhì)粒及

4、載體質(zhì)粒3種質(zhì)粒組成。包裝質(zhì)粒是HIV-1前病毒基因組5’端LTR由巨細胞病毒早期啟動子取代，3’LTR由SV40 polyA序列取代。包裝成分分別構(gòu)建在兩個質(zhì)粒上，一個表達gag和pol，另一個表達env。載體質(zhì)粒攜帶了5’端LTR，和全部5’端非翻譯區(qū)域，另外還帶有rev應(yīng)答元件（RRE）。包膜表達質(zhì)粒使用水皰性口炎病毒糖蛋白G基因(VSV-G)用來代替了原病毒的env基因。,5,,3.2第二代HIV-1來源的慢病毒載體

5、 1997年,Zufferey等將包裝質(zhì)粒上的vif、vpr、vpu和nef基因(即輔助基因)敲除,從而得到的,其他方面與第一代載體系統(tǒng)一致。,6,3.3第三代HIV-1來源的慢病毒載體 為減少復(fù)制型病毒的產(chǎn)生，可通過減少輔助質(zhì)粒與載體質(zhì)粒的同源性，或者將gag/ pol和rev編碼序列隔離,分散在不同的質(zhì)粒上。這樣的包裝系統(tǒng)由四質(zhì)粒代替原有的三質(zhì)粒包裝系統(tǒng)。 質(zhì)粒一攜帶了gag/ pol編碼序列及RRE ;

6、質(zhì)粒二包含了編碼rev的序列; 質(zhì)粒三是載體質(zhì)粒; 質(zhì)粒四表達env。,7,,3.4自身失活型（SIN）慢病毒載體 SIN載體的構(gòu)建是在原病毒載體基礎(chǔ)上刪除了病毒3’端LTR的U3區(qū)增強子和啟動子序列的片段。該區(qū)域出現(xiàn)突變則在HIV-1載體轉(zhuǎn)錄后，其5’LTR會因為缺失HIV-1所需要的啟動子和增強子序列而無法復(fù)制出完整長度的病毒基因組。,8,4.重組慢病毒的產(chǎn)生,常用瞬時轉(zhuǎn)染法，即將包膜質(zhì)粒，包裝質(zhì)粒和載體質(zhì)粒共轉(zhuǎn)染人

7、胚腎293T細胞直接產(chǎn)生生產(chǎn)細胞。最后慢病毒分泌到培養(yǎng)基中進行培養(yǎng)而得到大量載體慢病毒。,9,慢病毒在中樞神經(jīng)系統(tǒng)中的應(yīng)用,10,1. Introduction,Recombinant viral vectors have been used to study a variety of fundamental issues in developmental neurobiology, as well as pathogenesis

8、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

9、or reporter genes into central nervous system (CNS) cells in vivo and in vitro. 重組病毒載體已被用于研究各種發(fā)育神經(jīng)生物學(xué)中的基礎(chǔ)問題，以及各種神經(jīng)退行性疾病的發(fā)病機理和治療。慢病毒載體能轉(zhuǎn)導(dǎo)分裂的細胞，如神經(jīng)元，并介導(dǎo)中樞神經(jīng)系統(tǒng)（CNS）細胞在體內(nèi)和體外基因治療或報道基因而作為神經(jīng)生物學(xué)研究的有價值的工具。,11,1.1. Lentivir

10、al 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 D

11、NA 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. 慢病毒整合前復(fù)合物與核孔相互作用進入細胞核分裂的細胞，其中的前病毒DNA整合到宿主細胞的基因組DNA并進行主動運輸。

12、此功能是為什么慢病毒在有絲分裂后的細胞在中樞神經(jīng)系統(tǒng)的基因轉(zhuǎn)移載體的主要原因。,12,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

13、 deletions in the U3 region of the 3’ long terminal repeat (LTR) that are copied during reverse transcription to the 5’LTR. 自身失活型（SIN）慢病毒載體3’端LTR的U3區(qū)啟動子發(fā)生失活突變后，在逆轉(zhuǎn)錄過程中轉(zhuǎn)移至5’LTR。這樣的載體整合入靶細胞，將不會產(chǎn)生完整長度的載體RNA，因此命名為“自身

14、失活型載體”。,13,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

15、 of the proviral DNA and transduction efficiency in the brain. 為了增加基因在腦中的傳遞，新一代的慢病毒載體包含cPPT，一個來自gag區(qū)約180 bp的區(qū)域，從而增加了原病毒DNA進入核，也提高了在大腦中的轉(zhuǎn)導(dǎo)效率。,14,1.2. Targeted Gene Delivery in the CNS Using Pseudotyped Lentiv

16、iral 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

17、 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 glycoprot

18、ein (VSV-G). 慢病毒載體系統(tǒng)的另一個特征是攜帶病毒微粒的表面蛋白，包含HIV受體和共受體，從而改變或擴大的細胞類型的范圍，使得該載體可以結(jié)合并且進入。這個模型涉及取代的HIV-1包膜糖蛋白與另一種病毒的包膜糖蛋白，如皰疹性口腔炎病毒糖蛋白（VSV-G）。,15,1.3. Lentiviral Vector Production,The development of stable packaging cell

19、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

20、 stocks. 產(chǎn)生高滴度的慢病毒載體的穩(wěn)定的包裝細胞系的發(fā)展受到VSV-G表達的毒性的阻礙。因此，大多數(shù)人使用三質(zhì)粒系統(tǒng)。,16,Three plasmids are required: encoding the envelope glycoprotein (most commonly VSV-G), encoding the packaging proteins (minimally including gag

21、 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 LTR’s, the packaging signal , the

22、promoter and cDNA of interest and, in some cases, posttranslational regulatory elements and the central poly-purine tract (cPPT), which increase titer and expression.,17,2. Materials2.1. Transfection,1. A highly tran

23、sfectable 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.,18,6. High-quality plasmid DNA: transfer p

24、lasmid, packaging plasmid, envelope plasmid purified through cesium chloride/ethidium bromide equilibrium centrifugation or an anion-exchange matrix.7. Polybrene, 800 μg/mL stock solution in PBS.8. Large polyallomer

25、centrifuge tubes for concentrating the vector in a Beckman SW28 ul-tracentrifuge rotor.,19,2.2. Stereotactic Surgery,Anaesthesia mice.Surgical Preparation.Drilling and injection.Postoperative care.Perfusion.A manual

26、 for stereotaxic surgery such as Stereotaxic Surgery in the Rat.A mouse brain atlas such as The Mouse Brain in Stereotaxic Coordinates.,20,3. Methods,Transfection轉(zhuǎn)染Collection and Concentration of the Viral Supernat

27、ant 收集和集中的病毒上清Titering Lentiviral Vectors 滴定慢病毒載體,21,慢病毒在造血干細胞系統(tǒng)中的應(yīng)用,22,,造血干細胞（HSC）具有自我更新和分化為血液及免疫系統(tǒng)中各種成熟細胞的能力，許多HSC疾病如遺傳性、代謝性和感染性疾病或惡性腫瘤等有望通過基因治療的方法得到糾正。目的基因轉(zhuǎn)移HSC后，可隨著HSC的自我更新和分化在體內(nèi)長期表達，因此HSC是較理想的基因轉(zhuǎn)移靶細胞。,23,研究表明，VSV

28、-G假構(gòu)型HIV-I載體可不經(jīng)過對HSC的預(yù)刺激就能有效地將基因轉(zhuǎn)移到人早期干細胞并能在重度聯(lián)合免疫缺陷(SCID)鼠骨髓中穩(wěn)定地長期表達， Miyoshi等構(gòu)建VSV-G假構(gòu)型HIV-I載體，以綠色熒光蛋白（GFP）作為標記基因，將新鮮分離的人臍血CD34 +細胞在無血清及細胞因子培養(yǎng)基中轉(zhuǎn)染5小時，然后植入經(jīng)亞致死量照射的非肥胖型糖尿病/重度聯(lián)合免疫缺陷(NOD/SCID)鼠，可在受鼠脾臟、骨髓及外周血GFP的長期表達。,24,Me

29、thods,Isolation of Human CD34+ Hematopoietic Progenitor Cells 人CD34+造血祖細胞的分離Preparation of Lentiviral Vectors 慢病毒載體的制備Transduction of CD34+ Cells by Lentiviral Vectors 慢病毒載體轉(zhuǎn)導(dǎo)CD34+細胞Analysis of Transduce

30、d Human CD34+ Cells in NOD/SCID Mice分析轉(zhuǎn)人CD34+細胞的NOD/ SCID小鼠,25,慢病毒在眼科疾病治療中的應(yīng)用,26,1. Introduction,The primary aim of gene transfer into the retinal cells has been to investigate the developmental mechanisms of the reti

31、nal 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. 視網(wǎng)膜細胞基因轉(zhuǎn)導(dǎo)的主要目的是研究視

32、網(wǎng)膜細胞的發(fā)病機制或逆轉(zhuǎn)視網(wǎng)膜疾病。    目前，慢病毒和腺病毒載體在被用于研究和糾正的視網(wǎng)膜變性性疾病的基因治療。,27,Using an HIV vector carrying the green fluorescent protein (GFP) gene expressed from the cytomegalovirus (CMV) promoter, we showed that e

33、fficient 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載體攜帶綠

34、色熒光蛋白（GFP）基因其通過巨細胞病毒（CMV）啟動子的表達，可以在視網(wǎng)膜上得到高效和持久的基因的表達（圖1）。此外，基因表達被限制在感光體細胞中，是更有效的視紫紅質(zhì)子。,28,Fig. 1. Subretinal injection. 視網(wǎng)膜下注射 scleral approach.鞏膜方法(B) vitreous approach.玻璃體方法,29,2. Surgical Instruments,

35、Injection for the Small EyeVitrectomy for the Large Eye 大眼睛的玻璃體手術(shù)治療,30,Methods,Gene Delivery to the Subretinal Space of Rats and Mice大鼠和小鼠視網(wǎng)膜下腔的基因傳遞Gene Delivery to the Retina of Monkey 猴視網(wǎng)膜基因傳遞Gene Delivery to the