Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p''HR.cppt.3''1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.     

Production of retroviral vectors: review

Retroviral vectors are presently amongst the most widely used vectors in gene therapy clinical trials to target pathologies of different origins, such as cancers, genetic diseases or neurological disorders. This review provides an overview on the evolution of retroviral vector design and production for gene therapy applications, including state of the art developments in flexible producer cells and safe vectors. In addition, production and purification processes will be addressed, with a particular focus on the improvements undertaken to increase vector productivity and to reduce the rapid loss of infectivity, which presently represent the main challenges in retroviral vectors production for gene therapy.     

Downstream processing of cell culture-derived virus particles

Manufacturing of cell culture-derived virus particles for vaccination and gene therapy is a rapidly growing field in the biopharmaceutical industry. The process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. For the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. Owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs. For efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. Here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy.     

Effect of lentivirus-mediated shRNA targeting VEGFR-3 on proliferation, apoptosis and invasion of gastric cancer cells

It has been reported that vascular endothelial growth factor receptor 3 (VEGFR-3) is highly expressed in most tumor tissues, including gastric cancer. However, the effects of VEGFR-3 knockdown on the proliferation, apoptosis and invasion of gastric cancer cells and downstream signaling molecules have not yet been well established. In the present study, four short hairpin RNA (shRNA) sequences targeting the VEGFR-3 gene (NM_002020) were designed and cloned into a lentiviral vector, pRNAT-U6.2/Lenti, to construct four recombinant lentiviral vectors. The vectors with the two highest interfering efficiencies were selected to be co-transfected with packaging vectors in HEK293T cells to assemble lentivirus particles. Results from Western blot analysis showed that the VEGFR-3 shRNA-4 lentivirus-infected group (sh#4) had the highest efficiency of gene silencing in the MKN45 cell line compared with the parental and control group. The sh#4 group significantly slowed cell proliferation, decreased the mean percentage of S-phase cells and increased the mean percentage of G1 phase cells, promoted cell apoptosis, and also significantly inhibited cell invasion of MKN45 compared with the other two groups. Furthermore, the expression of the anti-apoptotic factor Bcl-2 was significantly decreased in the sh#4 group compared to that of the other two groups. Moreover, results from qRT-PCR revealed that knockdown of VEGFR-3 with the shRNA lentiviral vector resulted in down-regulation of the downstream neural cell adhesion molecule contactin-1 (CNTN-1). In conclusion, the recombinant lentivirus particles were able to remarkably suppress VEGFR-3 expression, regulate the cell cycle, inhibit proliferation and induce apoptosis in the MKN45 cell lines.     

Matching complementing functions of transformed cells with stable expression of selected viral genes for production of E1-deleted adenovirus vectors

Production of E1-deleted adenovirus (rAd) vectors requires complementation by E1A and E1B functions provided by the production cell line. The two cell lines most commonly used for production of rAd vectors, 293 and Per.C6, were derived from human primary cells and contain contiguous E1A and E1B sequences from the Ad genome. As an alternative system, we tested complementation of rAd vectors using sequential transfection of individual E1A and E1B expression cassettes into A549 human lung tumor cells, which support highly efficient replication of wild type adenovirus. We found that E1A function could be complemented in A549 cells by the mutant E1Adl01/07, and that E1B function could be provided in such cells using only the 55K E1B gene. Production yields in the resulting producer cell line, designated SL0003, were similar to those obtained from 293 cells without generation of detectable recombinant replication competent adenovirus.     

Optimized protocol for high-titer lentivirus production and transduction of primary fibroblasts

The lentivirus–short hairpin RNA (shRNA) system is a widely used tool for RNA interference. Multiple factors may affect the RNA interference efficiency during lentivirus production and transduction procedures. Thus, an optimized protocol is required to achieve high-titer lentivirus and efficient gene delivery. In the present study, lentivirus was produced by transfecting lentiviral transfer and packaging plasmids into HEK 293T cells. The factors affecting lentiviral titer were assessed, including lentiviral plasmid ratio, lentiviral transfer plasmid type, serum type for cell culture, transfection reagent–plasmid mixture incubation time, and the inoculation density of 293T cells for transfection. The high-titer lentivirus was achieved when plasmids were transfected at a molar ratio of 1:1:1:2, and the transfection reagent–plasmid mixture was replaced 6–8 h after transfection. The pLVX-shRNA2 lentiviral transfer plasmid was associated with the highest lentiviral titer, while both pLVX-shRNA2 and psi-LVRU6GP plasmids were associated with efficient RNA interference in target cells. The serum type for 293T cell culture affected the lentiviral titer significantly, while the inoculation density of 293T cells showed no influence on transfection efficiency or lentiviral titer. Moreover, the human primary fibroblasts infected with lentivirus, using the centrifugation method, achieved higher transduction efficiency than those infected with the non-centrifugation method. In conclusion, this study helped optimize lentiviral production and transduction procedures for more efficient gene delivery.     

Altering the tropism of lentiviral vectors through pseudotyping

The host range of retroviral vectors including lentiviral vectors can be expanded or altered by a process known as pseudotyping. Pseudotyped lentiviral vectors consist of vector particles bearing glycoproteins (GPs) derived from other enveloped viruses. Such particles possess the tropism of the virus from which the GP was derived. For example, to exploit the natural neural tropism of rabies virus, vectors designed to target the central nervous system have been pseudotyped using rabies virus-derived GPs. Among the first and still most widely used GPs for pseudotyping lentiviral vectors is the vesicular stomatitis virus GP (VSV-G), due to the very broad tropism and stability of the resulting pseudotypes. Pseudotypes involving VSV-G have become effectively the standard for evaluating the efficiency of other pseudotypes. This review samples a few of the more prominent examples from the ever-expanding list of published lentiviral pseudotypes, noting comparisons made with pseudotypes involving VSV-G in terms of titer, viral particle stability, toxicity, and host-cell specificity. Particular attention is paid to publications of successfully targeting a specific organ or cell types.     

一种新型慢病毒载体制备体系的改进

目的完善已初步建立的以痘病毒为辅助病毒的慢病毒载体（LV）瞬时制备体系。方法通过同源重组质粒pZIPPY-NEO/GUS,将vTF7-3痘病毒D13L基因的ORF通过同源重组被新霉素基因（neo）以及可视性筛选基因（GUS）替代,制备D13L缺陷性痘病毒（vTF7-3-ΔD13L）。结果经RT-PCR鉴定,D13L基因被完全敲除。制备体系中将vTF7-3-ΔD13L代替vTF7-3后,制备体系培养上清经RT-PCR、Western blotting分别检测到慢病毒载体特异性RNA序列以及特征性p24蛋白的存在,提示系统可以正常制备出慢病毒载体颗粒,并进一步评价了慢病毒载体的感染性。此外,对此系统的动力学特征也进行了初步分析。结论 vTF7-3-ΔD13L制备成功,通过此系统可制备出没有复制性痘病毒污染的慢病毒载体,本研究为该系统的大规模应用奠定了客观基础。     

Systematic improvement of lentivirus transduction protocols by antibody fragments fused to VSV-G as envelope glycoprotein

Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols and the limited activity of retronectin as LV enhancer, results in the application of a high multiplicity of infection (MOI) to achieve effective transduction efficiencies for a number of therapeutically relevant cells, e.g. CD34⁺ hematopoietic stem cells, T- and B-cells. Our study describes an optimized LV infection protocol including a non-toxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles, improving transduction efficiency at low MOI. Cell specificity of lentiviral vectors was increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system was validated with difficult to transduce human CD30⁺ lymphoma cells, and EGFR⁺ tumor cells. Highly efficient transduction of lymphoma cells was achieved, >50% of cells were transduced when MOI 1 was used. The scFv displaying lentiviral particles gained relative specificity for transduction of target cells. Preferential gene delivery to CD30⁺ or EGFR⁺ cells was increased 4-fold in mixed cell cultures by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation, poloxamer-based chemical adjuvant, and LV displaying scFv fragments increases transduction efficiencies of hard-to-transduce suspension lymphoma cells, and promises new chances for the future development of improved clinical protocols.     

Antibody-dependent gene transduction using gammaretroviral and lentiviral vectors pseudotyped with chimeric vesicular stomatitis virus glycoprotein

Gammaretroviral and lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) have been used for stable gene transfer because of their broad host range and high mechanical strength. In the present study, an expression plasmid for chimeric VSV-G, consisting of a ZZ fragment derived from Staphylococcal protein A fused to the N-terminus of VSV-G (ZZ-VSV-G), was constructed to produce viral vectors capable of antibody-dependent gene transduction. Gammaretroviral (based on mouse stem cell virus, MSCV) and lentiviral (based on human immunodeficiency virus type 1, HIV-1) vectors pseudotyped with ZZ-VSV-G were produced without the loss of antibody-binding activity. The production of infectious viral particles was promoted by the addition of an expression plasmid for native VSV-G and antibody-dependent gene transduction was achieved using plates coated with antibodies. This system may be useful for the genetic transduction of cells expressing specific proteins on their surface, and for screening of antibodies specific for cell surface receptors.     

人心肌肌球蛋白轻链基因启动子驱动的GFP和Luc报告基因在人心肌细胞系中的表达

目的:构建人肌球蛋白2v基因启动子(pMLC2v)驱动的绿色荧光蛋白(GFP)和萤光素酶(Luc)报告基因慢病毒载体并观察其在人心肌细胞系(HCM)和人肺癌细胞株A549中的整合表达特征。方法:应用去毒化的人类I型免疫缺陷病毒和pMLC2v-GFP或pMLC2v-Luc报告基因构建慢病毒示踪载体,转染HCM和A549细胞,激光共聚焦显微镜及生物发光检测仪观察2种报告基因在不同细胞生长进程中的表达特征。非特异性启动子驱动的GFP(GFP_C)和红色荧光蛋白(RFP_C)报告基因作为对照。结果:2种细胞转染GFP_C和RFP_C后第3 d,都表达GFP和RFP;而HCM只在转染pMLC2v-GFP和pMLC2v-Luc 21 d后表达GFP和Luc,A549细胞不表达。结论:pMLC2v主要在培养21 d后新增殖的人心肌细胞中驱动GFP和Luc报告基因表达,这为监测干细胞向心肌细胞的分化进程提供了可靠的病理及活体示踪工具。     

TCAB1基因RNAi慢病毒载体的构建及在人子宫内膜间质细胞中沉默TCAB1基因的效应

目的构建TCAB1基因RNAi慢病毒载体,为子宫内膜异位症(EMs)的基因靶向治疗提供理论依据。方法化学合成3条靶向TCAB1基因的siRNA,转染HEK-293T细胞,24h后采用荧光素酶报告系统筛选有效siRNA,其对TCAB1mRNA有明显抑制作用。针对已经筛选确定的TCAB1基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经XhoI和HpaI酶切后的LV1载体[含U6启动子和绿色荧光蛋白(GFP)]连接产生LV1-shRNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。用LV1-shRNA、pRev、pVsvg、pcgv四质粒共转染包装细胞HEK-293T细胞,包装产生慢病毒,分别于转染后48h、72h收取病毒上清,感染人子宫内膜间质细胞(ESC),进行RT-qPCR检测TCAB1基因相对表达量,检测LV1-shRNA干扰效率。结果 PCR和测序证实,成功构建TCAB1 shRNA的慢病毒载体LV1-shRNA。结论成功构建TCAB1基因的RNAi慢病毒载体,其转染ESC细胞后显著抑制了TCAB1的表达,为子宫内膜异位症的基因靶向治疗提供了实验基础。     

Nanog慢病毒载体构建及在胚胎干细胞分化调控中的应用

BACKGROUND:There is a lack of safe and effective modular lentivectors in differentiation regulation of embryonic stem cells. OBJECTIVE:To construct a modular lentivector, Puro-Nanog-hrGFP, with Nanog promoter-controlled humanized renilla reniformis green fluorescent protein (hrGFP) and puromycin and to observe the expression of Nanog during the differentiation of Nanog-hrGFP-transfected mouse embryonic stem cell lines. METHODS:After PCR amplification, Nanog, hrGFP and entry vector were recombined into the pDest-puro vectors to generate the lentiviral expression vector, Puro-Nanog-hrGFP, by the LR reaction. Through lentivirus production, transduction and puromycin screening, the transduced cell lines with Nanog-hrGFP gene were generated and identified. Expression of Nanog during the differentiation of transgenic mouse cell lines was detected. RESULTS AND CONCLUSION:The lentiviral expression vectors Puro-Nanog-hrGFP were constructed successfully by Gateway technology, and then the transducted cell lines were obtained by lentiviral infection. The expression of Nanog was gradually decreased during the process of transgenic cell lines differentiation, which provides a new tool for further investigation on regulation of stem cell differentiation.     

Strategies for retargeted gene delivery using vectors derived from lentiviruses

With the development of the first viral vector systems 20 years ago [Mann et al., 1983; Watanabe and Temin, 1983] gene therapy strategies have come to the forefront of novel therapeutics [Cavazzana-Calvo et al., 2000]. A deeper understanding of vector biology and the molecular mechanisms of disease alongside tremendous advances in vector technology have significantly advanced the field of human gene therapy. Over the last few years several challenges needed to be overcome in order to bring gene therapy strategies closer to the clinic. These hurdles include the preparation of large amounts of stable, high titre vectors, minimising vector-related immunology and last but not least targeting infection and transgene expression to tissue or cells, which in many cases are not or only slowly dividing. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Moreover, lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle thus opening up the possibility to target non-dividing target cells and tissues. Human immunodeficiency virus (HIV) based vectors have been used in vitro and in vivo in a number of situations, however, safety concerns still exist, and therefore the development of vector systems based on primate as well as non-primate lentiviruses is ongoing. Concomitantly with lentiviral vector design, much has been learned about the incorporation of heterologous env proteins on lentiviral cores in order to combine specific targeting properties of envelope glycoproteins with the biological properties of lentiviral vectors. In this review article we will give an overview over advantages lentiviral vector systems offer. We will then discuss the current state of our understanding of the structure and function of viral envelope glycoproteins and emerging targeting strategies based on retroviral and lentiviral vector systems.     

Adenovirus vector production and purification

Replication deficient adenovirus vectors are frequently used tools for the delivery of transgenes in vitro and in vivo. In addition, several therapeutic products based on adenovirus are under clinical development. This review outlines adenovirus vector production discussing different vector types, available production cell lines and state of the art of production process development and purification.     

Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre

We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence calledloxP. Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters. The resulting 293 Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive “molecular switch” High efficiency recombination was observed for Ad viral DNA containingloxP sites. The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination betweenloxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined byloxP sites in viral genomes.     

金黄色葡萄球菌肠毒素C3 过表达慢病毒载体的构建及鉴定

目的:构建金黄色葡萄球菌肠毒素C3(SEC3)过表达慢病毒载体并体外检测其表达目的基因。方法:聚合酶链反应(PCR)扩增SEC3 基因片段;用AgeI 酶切线性化GV365 慢病毒载体,通过连接反应构建GV365-SEC3 载体,运用PCR方法鉴定阳性克隆载体;转染293T 细胞包装慢病毒,观察细胞荧光及Western blot 检测慢病毒载体表达,HIV-1 p24 ELISA 法测定慢病毒载体滴度。结果:获得目的基因,并成功构建GV365-SEC3 慢病毒载体,通过PCR 及DNA 测序鉴定,证明GV365-SEC3 质粒构建正确;转染293T 细胞后可观察到大量荧光细胞,经蛋白电泳得到29 kD 大小的蛋白条带,与目的基因蛋白相符合,ELISA 检测病毒载体滴度为5 108 TU/ ml。结论:成功构建GV365鄄SEC3 过表达慢病毒载体,为后期研究其体内外对抗肿瘤的作用与机制奠定基础。     

Beclin1 基因慢病毒表达载体的构建和鉴定

背景：Beclin1基因是哺乳动物的自噬调控基因。 目的：实验拟构建Beclin1 基因慢病毒过表达载体。 方法：聚合酶链反应扩增目的基因Beclin1 后插入慢病毒表达载体pLenex中,构建重组载体pLenex-Beclin1。使用聚合酶链反应、双酶切和DNA的测序方法对其进行鉴定,并与辅助包装质粒共感染293T细胞。慢病毒颗粒转染非小细胞肺癌A549细胞后,用蛋白质印迹法检测Beclin1 基因的过表达效率。 结果与结论：聚合酶链反应鉴定结果显示扩增的阳性片段已插入pLenex载体,聚合酶链反应、双酶切和DNA测序结果表明,重组慢病毒载体pLenex-Beclin1 的插入序列完全正确,重组慢病毒载体感染A549细胞后,细胞内Beclin1蛋白高效表达。结果证实,实验成功构建了Beclin1 基因慢病毒过表达载体。     

中国版纳猪MHCI类P1分子全长的原核表达与纯化

目的：获得原核表达的中国版纳猪ＳＬＡＩ类Ｐ１蛋白质分子。方法：ＰＣＲ扩增去信号肽的ＳＬＡＩ类Ｐ１ｃＤＮＡ序列，亚克隆至ｐＧＥＭＴ载体，测序。将亚克隆的Ｐ１　ｃＤＮＡ片段插入表达载体ｐＥＴ４２ｂ（＋），构建重组表达质粒ｐＥＴ－４２ｂ（＋）／ｓｌａ－ｐｌ，转化Ｅ·ｃｏｌｉ表达菌 ＢＬ２１－ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＬ，ＩＰＴＧ诱导 Ｐ１－８ ｘ ｈｉｓ融合蛋白表达，经包涵体洗涤，８ ｍｏｌ／Ｌ尿素变性溶解，Ｎｉ２＋亲和层析，梯度透析后，定量保存。ＳＤＳ－ＰＡＧＥ、ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ鉴定目的蛋白的表达与纯化。结果：目的蛋白（分子量３９．５ ｋＤ）表达量占细菌总蛋白 １５％，每升表达菌获得纯度９５％的目的蛋白 ４０ ｍｇ～６０ｍｇ。结论：成功建立猪 ＳＬＡ分子全长原核表达、纯化体系，为建立间接识别猪移植抗原ＳＬＡＩ类分子的人Ｔ细胞系及表位分析打下基础。