Gene transfer into the central nervous system in vivo using a recombinanat lentivirus vector

Gene transfer vectors derived from human immunodeficiency virus (HIV-1) efficiently transduce nondividing cells and may provide for the delivery of their gene products to discrete regions of the brain. We investigated whether stable gene transduction can be achieved in cells of the central nervous system (CNS) in vivo by a potent lentivirus vector. The herpes simplex virus type 1 protein VP22 has been known to facilitate intercellular protein transport and thereby provides an opportunity to increase the effectiveness of therapeutic genes by enhancing the delivery of their protein products. We developed a lentiviral vector construct expressing enhanced green fluorescent protein (EGFP) fused at its N-terminus to the herpes simplex virus VP22. In order to determine expression of the fusion protein in specific cells such as neurons in the CNS, a neuron-specific promoter was also placed into the construct. The viral vectors were injected directly into the striatum and hippocampus of mouse brains. We found that the lentivirus vector efficiently and stably transduced nondividing cells in the CNS with transgene expression for over 3 months. We also show that the delivery of VP22-EGFP fusion protein encoded by the lentivirus was effectively transported between neuronal cells via axons in vivo. Doubly labeled experiments revealed that our lentiviral vector is capable of delivering gene products to neurons and astrocytes in CNS. The data also demonstrate that up to 90% of the CNS cells transduced by our lentiviral vector under the control of the neuronal promoter are neurons.     

Lentivector-mediated delivery of GDNF protects complex motor functions relevant to human Parkinsonism in a rat lesion model

Although viral vector-mediated delivery of glial cell-line derived neurotrophic factor (GDNF) to the brain has considerable potential as a neuroprotective strategy in Parkinson's disease (PD), its ability to protect complex motor functions relevant to the human condition has yet to be established. In this study, we used an operant task that assesses the selection, initiation and execution of lateralized nose-pokes in Lister Hooded rats to assess the efficacy with which complex behaviours are protected against neurotoxic lesions by prior injection of a lentiviral vector expressing GDNF. Unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) caused rats to attempt fewer trials and to make more procedural errors. Lesioned rats also developed a pronounced ipsilateral bias, with a corresponding drop in contralateral accuracy. They were also slower to react to contralateral stimuli and to execute movements bilaterally. Rats that were pre-treated 4 weeks prior to lesion surgery with an equine infectious anaemia virus (EIAV) vector carrying GDNF [EIAV-GDNF, injected into the striatum and above the substantia nigra (SN)] performed significantly better on all of these parameters than control rats. In addition to the operant task, EIAV-GDNF successfully rescued contralateral impairments in the corridor, staircase, stepping and cylinder tasks, and prevented drug-induced rotational asymmetry. This study confirms that GDNF can protect against 6-OHDA-induced impairments in complex as well as simple behaviours, and reinforces the use of EIAV-based vectors for the treatment of PD.     

CD81-induced behavioural changes during chronic cocaine administration: in vivo gene delivery with regulatable lentivirus

CD81, a tetraspanin transmembrane protein involved in cell adhesion, is up-regulated in the mesolimbic dopaminergic pathway 24 h following acute administration of high doses of cocaine [Brenz-Verca et al., (2001) Mol. Cell. Neurosci., 17, 303-316]. Further evidence consecutive with this observation and based on microarray analysis are presented here. In addition, a regulatable lentivirus was developed bearing the rat CD81 gene under the control of a tetracycline inducible system. This lentivirus vector was stereotaxically injected into the ventral tegmental area (VTA) of two groups of animals, one fed water (expressing CD81) and the other Doxycycline solution (which down-regulates CD81 expression) and locomotor activity after chronic cocaine administration (10 mg/kg daily) was monitored. After 2 weeks, the groups were inverted, animals receiving water were placed on Doxycycline and the second group was placed on water. In all cases highly a significant increase (3.2-fold) in locomotor activity was observed in animals expressing CD81 in the VTA vs. animals placed on Doxycycline. Similar studies where CD81 was delivered into the nucleus accumbens (NAcc) resulted in significantly higher effects (30%), in accordance with microarray data and our previous reports, yielding a 4.2-fold increase in locomotor activity. No change was observed under similar conditions in control animals, which were injected a regulatable lentivirus expressing GFP. These findings suggest that CD81 expression in the mesolimbic dopaminergic pathway contributes to behavioural changes associated with cocaine sensitization. This study provides a powerful approach for evaluating a gene function in vivo in a single animal under various paradigms, even on gene candidates, which display small changes of expression.     

Evidence for Recombination in the Envelope Gene of Maedi-visna Virus

Frequent recombination occurs during replication in all retroviruses examined. This increases the genetic variation in the retroviral population and may be of importance in the evolution of the virus. Maedi-visna virus (MVV), a retrovirus of sheep, has a highly variable envelope gene. In a previous experiment, 20 sheep were infected with an uncloned strain of MVV and virus was isolated at regular intervals for 7 years. We sequenced the envelope genes of a number of these strains and found evidence for recombination that may have contributed to the observed high frequency of antigenic variants.     

携带Bcl-xl基因的重组慢病毒的制备和鉴定

目的制备携带Bcl-xl基因的重组慢病毒,并鉴定其感染细胞有效性。方法从本所以前构建的重组质粒pAAV-Bcl-xl获得Bcl-xl基因的编码序列,将其置换掉慢病毒质粒pSin-EF2-SOX2-Pur中的SOX2基因编码序列,从而得到重组慢病毒质粒pSin-EF2-Bcl-xl-Pur。然后利用这一质粒包装制备了携带Bcl-xl基因的重组慢病毒颗粒,用其感染细胞后用Western blot和流式检测仪分析了感染前后细胞中Bcl-xl基因的表达情况。结果感染后细胞中Bcl-xl蛋白的表达水平显著高于感染前。结论携带Bcl-xl基因的重组慢病毒颗粒包装成功,能高效感染细胞,为后续的遗传改造树突细胞打下了基础。     

稳定过表达生长分化因子５基因大鼠脂肪干细胞系的建立

目的:探讨慢病毒介导的稳定过表达生长分化因子5(GDF-5)基因大鼠脂肪干细胞(ASCs)的构建条件和方法。方法:取大鼠腹股沟脂肪垫组织,采用Ⅰ型胶原酶消化贴壁法分离培养大鼠ASCs。倒置相差显微镜观察细胞形态,CCK-8法测定细胞生长曲线,流式细胞仪鉴定细胞表型。制备带GDF-5/GFP融合基因的慢病毒载体系统,探索不同感染复数(MOI=1、5、10、20、40、60、80、100)的转染效率,选择最佳MOI,采用流式细胞仪检测转染效率。对转染细胞行流式细胞筛选,测定筛选后转染细胞的阳性率。筛选出的细胞行细胞爬片,DAPI染色,形态学上进一步验证细胞阳性率;并采用CCK-8法检测转染后细胞活力。结果:成功培养大鼠ASCs,流式细胞免疫表型鉴定:间充质干细胞表面抗原(CD90、CD29、CD44、CD105)表达阳性,造血细胞表面抗原(CD45、CD34)和骨髓干细胞表面抗原(CD106)表达阴性。成功构建GDF-5过表达慢病毒载体系统,慢病毒转染大鼠ASCs最佳MOI为40,转染率为65%。采用GFP荧光流式细胞筛选技术筛选后阳性率可提高至96%。CCK-8法显示,转染后细胞活力、生长曲线与未转染细胞无明显差异。结论:胶原酶消化法可成功培养大鼠ASCs,流式细胞筛选技术可显著提高转染细胞阳性率,且对细胞系活力无显著影响。     

Characterization and gene transfer in mesenchymal stem cells derived from human umbilical-cord blood

It has been shown that the stromal-cell population found in bone marrow can be expanded and differentiated into cells with the phenotypes of bone, cartilage, muscle, neural, and fat cells. However, whether mesenchymal stem cells (MSCs) are present in human umbilical-cord blood (UCB) has been the subject of ongoing debate. In this study, we report on a population of fibroblastlike cells derived from the mononuclear fraction of human UCB with osteogenic and adipogenic potential, as well as the presence of a subset of cells that have been maintained in continuous culture for more than 6 months. These cells were found to express CD29, CD44, CD90, CD95, CD105, CD166, and MHC class, but not CD14, CD34, CD40, CD45, CD80, CD86, CD117, CD152, or MHC class II. We also compared gene expression after gene transfer using lenti- and adenoviral vectors carrying the green fluorescence protein to the MSCs derived from UCB because a reliable gene-delivery system is required to transfer target genes into MSCs, which have attracted attention as potential platforms for the systemic delivery of therapeutic genes. The lentiviral vectors can transduce these cells more efficiently than can adenoviral vectors, and we maintained transgene expression for at least 5 weeks. This is the first report showing that UCB-derived MSCs can express exogenous genes by way of a lentivirus vector. These results demonstrate that human UCB is a source of mesenchymal progenitors and may be used in cell transplantation and a wide range of gene-therapy treatments.     

pLenti6/V5-DEST-TAZ载体的构建及其对成骨前体细胞MC3T3-E1分化的影响

目的：构建人真核表达的TAZ慢病毒载体pLenti6/V5-DEST-TAZ,转染293FT 细胞获得重组慢病毒颗粒,探讨TAZ对成骨前体细胞MC3T3-E1 分化的调控作用。方法：将已经过测序验证的含有TAZ基因慢病毒入门质粒pENTR(tm)221-TAZ通过LR反应克隆到慢病毒载体pLenti6/V5-DEST中。对重组质粒进行酶切鉴定,并在细胞中验证表达。将该重组载体和其他PackingMix 3 个质粒载体充分混合,用阳离子脂质体转染293FT 细胞,培养和待细胞完全裂解后收集富含TAZ基因的病毒颗粒上清液,取适量上清液感染MC3T3-E1细胞,采用杀稻瘟菌素Blastcidin 筛选,建立稳定过量表达 TAZ 蛋白的 MC3T3-E1/TAZ细胞系。用条件培养基诱导 MC3T3-E1和 MC3T3-E1/TAZ细胞系向成骨细胞定向分化,von Kossa 和茜素红染色观察MC3T3-E1和MC3T3-E1/TAZ细胞的成骨分化。结果：凝胶电泳和测序结果均证明TAZ重组慢病毒载体pLenti6/V5-DEST-TAZ构建正确,并能在细胞中正确表达。与辅助质粒共转包装细胞获得慢病毒颗粒,并成功感染MC3T3-E1细胞。Von Kossa 染色和茜素红染色, MC3T3-E1/TAZ细胞系中生成的磷酸钙比MC3T3-E1细胞系中生成的磷酸钙多。结论：成功构建了人真核表达的质粒载体pLenti6/V5-DEST-TAZ,并能在细胞中正确表达;成功包装了TAZ病毒,并获得过表达TAZ的MC3T3-E1细胞;过表达TAZ促进MC3T3-E1向成骨细胞分化。     

APE1基因RNAi慢病毒载体的构建与鉴定

目的构建APE1基因慢病毒载体,为其后续的体内外实验研究提供基础。方法应用基因工程技术,筛选出两条针对APE1基因的RNAi靶序列KD1、KD2,分别与pGCIL-GFP载体连接,经转化筛选鉴定后,包装产生慢病毒颗粒并测定病毒滴度,分别命名为LV-APE1-shRNA1、LV-APE1-shRNA2,两种病毒颗粒分别感染MHCC97-H细胞,设为感染LV-APE1-shRNA1、LV-APE1-shRNA2细胞组,同时设未感染病毒组和感染空载体细胞组。Real-time PCR和Western blot检测MHCC97-H细胞中APE1基因的mRNA和蛋白的表达。结果 PCR及测序结果与预期结果一致,LV-APE1-shRNA1、LV-APE1-shRNA2的病毒滴度为4×10^8TU/mL和7×10^8TU/mL。与未感染病毒组和感染空载体细胞组相比,2组慢病毒组APE1基因mRNA和蛋白的表达均明显下降,LV-APE1-shRNA1、LV-APE1-shRNA2对APE1基因的mRNA表达的抑制率分别为75%,90%,对APE1蛋白表达的抑制率达90%,95%。结论成功构建高效阻断APE1基因表达的RNAi慢病毒表达载体,为应用RNAi进一步研究APE1基因在肝癌中的作用机制和基因治疗奠定基础。     

人TNF-α重组慢病毒载体构建及其脐血间质干细胞表达

目的构建带有人TNF-α基因的慢病毒载体,并且观察该基因在体外人脐血间质干细胞中的表达。方法通过逆转录聚合酶链反应(RT-PCR)从PCD DNA-TNF-α质粒中获得人TNF-α基因,利用Infusion技术重组构建慢病毒载体穿梭质粒pGC-FU-TNF-α,在脂质体Lipofectamine 2000介导下与结构质粒pHelper 1.0和包膜质粒pHelper 2.0共转染293T细胞包装生产慢病毒。将人脐血间质干细胞分为实验组(pGC-FU-TNF-α)、空载体对照组(pGC-FU-EGFP)及空白组(脐血间质干细胞),分别用重组慢病毒、空载慢病毒、PBS感染后,采用RT-PCR以及ELISA方法检测TNF-α表达。结果所获TNF-α基因测序证明与GenBank中序列一致;重组慢病毒载体质粒pGC-FU-TNF-α经鉴定正确;三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为2×107TU/L,感染脐血间质干细胞后RT-PCR、ELISA检测3组细胞均有TNF-α表达,其中实验组大量表达TNF-α,与其余两组比较差异有显著性(F=11.677、21.321,P<0.01)。结论成功构建带有TNF-α基因的慢病毒载体并实现在脐血间质干细胞中的表达,为脐血间质干细胞转基因治疗胃癌的应用奠定基础。