High efficiency and long-term foreign gene expression in cultured liver sinusoidal endothelial cells by retroviral transduction.

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.     

重组小鼠干细胞逆转录病毒载体高效基因转染CD41+、UT7、U937和MDA-MB-435细胞

目的:研究重组小鼠干细胞逆转录病毒载体介导基因转染,探索一条高效基因转染的途径,为重组小鼠干细胞逆转录病毒载体在基因转染中的应用提供理论依据和奠定实验基础。方法:①逆转录病毒载体的构建:EC1-4(repeats1-4ofcadherin-5extracellulardomains)基因克隆产物和mutant(Ser222A)MEK1基因克隆产物,Bg1Ⅱ和EcoRⅠ限制性内切核酸酶切割后,克隆进入逆转录病毒表达载体pMSCV。②CD41⁺细胞的获取和细胞培养:从脐带血分离的CD34⁺细胞通过TPO诱导表达CD41,FACS分离CD41⁺细胞。高糖DMEM培养液培养NIH3T3和MDA-MB-435细胞,U937细胞培养在RPMI-1640培养液,UT7细胞是细胞因子依赖性细胞株,Iscove'smodifiedDulbeco's培养液中加入GM-CSF。③测定病毒滴度:逆转录病毒载体转入包装细胞293,36h后收集病毒上清液,感染NIH3T3细胞,流式细胞仪测定病毒滴度。④Westernblot:基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,Westernblot检测基因产物的表达。结果:293细胞产生高滴度MEK1pMSCV病毒:3.1×10⁷,高滴度EC1-4pMSCV病毒:1.0×10⁸。用稀释8倍的病毒转染基因,重组逆转录病毒MEK1pMSCV转染白血病细胞株UT7和U973,GFP阳性细胞(转染阳性细胞)分别是60.73％、72.56％。重组逆转录病毒MEK1pMSCV转染原代培养细胞CD41⁺,GFP阳性细胞为30.57％。重组逆转录病毒EC1-4pMSCV转染人乳腺癌细胞株MDA-MB-435,GFP阳性细胞为97.54％。TPO作用CD41⁺和UT7细胞以及血清对U973细胞的作用,显示出外源mutationMEK基因的dominantnegative的效应,实验组磷酸化的MEK1减少。EC1-4基因转染的MDA-MB-435细胞表达了EC1-4基因产物。结论:重组小鼠干细胞逆转录病毒载体能高效基因转染CD41⁺、UT7、U937和MDA-MB-435细胞,转染的基因能稳定地表达。     

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.     

Engineering analysis of ex vivo retroviral transduction system

The overall dynamics of the retrovirus-cell encounter under a static retroviral transduction system can be described in terms of the process of uptake (adsorption/internalization), decay, and diffusion. In this study, a mathematical model illustrating these processes was derived assuming a semi-infinite domain and solved analytically using the Laplace transform. The closed-form solutions for retroviral concentrations and time course profile of transduced cell colonies are presented to clarify the contributions of the processes involved in the retroviral transduction system. To manifest the usefulness of the closed-form solutions, the neomycin-resistant gene encoding retroviruses produced by two different packaging cells (human 293 cells and murine GP+E86/LNCX cells) were employed to transduce NIH 3T3 cells, which formed neomycin-resistant colonies after G418 selection. The experimental results were curve fitted with the model-derived analytical solutions to quantitatively determine transduction rate constant k and initial concentration of infectious retrovirus C₀. Our study showed that the vesicular stomatitis virus G protein pseudotyped retrovirus produced from 293 packaging cells exhibited much higher transduction rate (k=0.0480cm/h} than the ecotropic retrovirus (k=0.0102 cm/h) produced from GP+E86/LNCX cells. The fitted values of C₀ are approximately two orders of magnitude higher than the experimentally estimated titers for both retroviruses. © 2002 Biomedical Engineering Society. PAC2002: 8717Jj, 8718Ed, 8716Sr     

小鼠IL-3 cDNA逆转录病毒载体的构建及在NIH3T3细胞中的表达

为了探讨放射损伤小鼠IL-3基因治疗可能性,本文作者将小鼠IL-3cDNA亚克隆到逆转录病毒载体pLXSN上。将重组的逆转录病毒载体用电穿孔法和磷酸钙共沉淀法转染双向包装细胞系PA317,经新霉素类抗菌素G418选择,得到了多个产病毒细胞克隆。用含有复制缺陷病毒的产病毒细胞培养上清转化NIH3T3细胞,得到了数个自发分泌IL-3的克隆。在去掉G418,传代15代,约2个月后,这些克隆分泌IL-3的能力没有明显的变化。     

Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration

Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.     

Characterization of recombination events leading to the production of an ecotropic replication-competent retrovirus in a GP+envAM12-derived producer cell line

Replication-competent retrovirus (RCR) was identified in a GP+envAM12-derived producer cell, containing the MFG-S-Neo retroviral vector, using a marker rescue assay. Studies were undertaken to determine the origin and structure of this RCR. Receptor interference assays demonstrated that the virus was pseudotyped with an ecotropic envelope. Molecular analysis demonstrated the presence of a MoMLV ecotropic env recombinant where the neomycin resistance gene of the MFG-S-Neo vector was replaced by MoMLV ecotropic env. Additional recombinants linking the retroviral pol gene to neo and the neo gene to MoMLV env were also identified. A full-length MoMLV retroviral genome was detected by nested PCR in the contaminated amphotropic producer cells and in cells infected with its supernatant. Unexpectedly, this was also present in the GP+E86 packaging cells together with a previously undescribed envelope construct possessing a full 5' and 3' LTR, although these cells were consistently negative for the presence of RCR. These anomalies in the GP+E86 packaging cell line result in increased homology with the MFG-S-Neo vector, leading to an increased risk for the production of RCR. Our findings point to a need for increased vigilance when using these packaging lines to generate replication-defective retrovirus.     

含人组织型纤溶酶原激活剂cDNA重组逆转录病毒的制备…

A recombinant retroviral vector containing tissue-type plasminogen activator (t-PA) cDNA was constructed and transfected into PA317 viral packaging cells, forming intact virus particles. Under electron microscope the recombinant retroviral particles were composed of envelope, capsid and core. These viral particles were spherical with a diameter of 90-180nm, and spread dispersely in the cells. NIH3T3 cell infected by retrovirus particles were screened with G418. The virus titer of 6 x 10(8) CFU/L was verified by counting the positive clones two weeks after screening. The expression of t-PA was demonstrated in the NIH3T3 cells infected with the recombinant virus.     

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.     

乙型肝炎病毒C基因重组逆转录病毒的构建和在真核细胞的表达

目的　构建含HBVC基因的重组逆转录病毒以用于慢性乙型肝炎的免疫治疗。方法　用PCR法扩增HBV的C基因片段 ,定向插入逆转录病毒质粒载体pLXSN中 ,重组质粒用脂质体转染包装细胞PT67,G418筛选抗性细胞 ,用抗性细胞培养上清液中的重组病毒感染NIH3T3、EL4和鼠骨髓来源的树突状细胞 (DC) ,用PCR检测目的基因的插入 ,用间接免疫荧光和流式细胞仪检测目的基因的表达 ,基因修饰的DC免疫C57BL 6鼠观察其诱导细胞免疫的能力。结果　含HBVC基因重组质粒载体经PCR、酶切和序列分析鉴定为阳性 ,转染PT67后经G418筛选获得抗性细胞 ,抗性细胞培养上清液中含有重组逆转录病毒 ,病毒效价达 3× 10 5CFU ml,感染NIH3T3和EL4细胞后PCR鉴定含目的基因 ,间接免疫荧光、流式细胞仪鉴定有HBcAg表达 ,基因修饰后的DC免疫C57BL 6鼠能诱导特异性CTL应答。结论　构建的HBVC基因重组逆转录病毒可将目的基因转移至真核细胞并得到有效表达     

重组跨膜型人CD55分子在小鼠NIH3T3细胞上的表达及其补体溶破保护功能的研究

目的 :在小鼠NIH3T3细胞转染表达人天然GPI锚固型CD5 5和重组跨膜型CD5 5 TM分子 ,观察比较它们对人补体溶破异源细胞的抑制功能。方法 :将带有CD5 5cDNA、CD5 5 TMcDNA的重组逆病毒表达质粒CD5 5 pLXSN、CD5 5TM pLXSN经脂质体法转染PA317细胞 ,用病毒上清感染小鼠成纤维母细胞NIH3T3。经G418加压筛选 ,利用FACS检测获得表达CD5 5和CD5 5 TM分子的阳性细胞克隆 ,通过MTT比色法比较两种分子对人血清补体溶破细胞的抑制功能有无差别。结果 :细胞转染筛选获得多个表达跨膜型人CD5 5分子的NIH3T3细胞克隆 ,补体杀伤试验证实其具有抑制人补体溶破的功能 ,且两种分子的补体抑制功能无明显差异。结论 :成功地建立了稳定表达天然CD5 5、跨膜型CD5 5分子的小鼠NIH3T3细胞 ,证实其表达的GPI型CD5 5分子和CD5 5TM分子均具有抑制人补体溶破细胞的功能 ,为进一步探讨应用跨膜型的CD5 5分子对PNH进行基因治疗奠定了基础。     

Enhancement of gene transduction efficiency in cancer cells using cationic liposome with hyperthermia

We evaluated the effects of hyperthermia on the efficiency of gene transduction by using a cationic liposome to develop an efficient method for lipofection. We used Lewis lung carcinoma (LLC), NIH3T3, and A549 cell lines, with Lipofectamine reagent as the cationic liposome and the LacZ gene as the reporter gene. In LLC, co-incubation of the cationic liposome and plasmid DNA complex (lipoplex) with the cells for 2 h at 41 degrees C enhanced the efficiency of gene transduction approximately 1.4-fold compared to incubation for 2 h at 37 degrees C, as measured by X-gal staining and beta-galactosidase activity. In cell lines NIH3T3 and A549, the efficiency of gene transduction showed a tendency toward enhancement after 2 h co-incubation with lipoplex at 41 degrees C compared to that at 37 degrees C, as measured by X-gal staining. This is the first study to demonstrate the enhancement of gene transduction efficiency achieved by using a cationic liposome under conditions of hyperthermia. This method should prove useful for lipofection in other cancer cells.     

Characterization of recombinant helper retroviruses from moloney-based vectors in ecotropic and amphotropic packaging cell lines

We have characterized the recombinant replication-competent retrovirus (RCV) arising from p delta N2-derived vectors in the packaging cell lines psi 2 (ecotropic) and PA317 (amphotropic). Detailed restriction patterns and sequence of the envelope region of these RCVs has indicated that they arose from recombination events between the virus plasmids used to create the packaging cell line and the vectors. There was no evidence of recombination involving endogenous murine retroviral sequences in the packaging cell line or in transduced hematopoietic cells. In addition, we have confirmed that the mutation of the start codon of the pXM5(N2) derivatives gag+ sequence drastically decreased the occurrence of RCV production. These results offer encouragement that the risk of RCV production can be adequately decreased in gene therapy applications of defective retrovirus vectors.     

Genomic characterization of a highly oncogenic env gene recombinant between amphotropic retrovirus of wild mouse and endogenous xenotropic virus of NIH swiss mouse

A highly oncogenic retrovirus (strain 10A/1) recovered from NIH Swiss mice inoculated with wild mouse amphotropic virus (strain 1504A) was studied by T1-oligonucleotide fingerprinting of the RNA genome. The virus was found to be an env gene recombinant between the parental 1504A and endogenous AT124 or related xenotropic virogenes of NIH Swiss mice.     

Multidrug resistance and retroviral transduction potential in human small cell lung cancer cell lines.

Multidrug resistance (MDR) remains a major problem in the successful treatment of small cell lung cancer (SCLC). New treatment strategies are needed, such as gene therapy specifically targeting the MDR cells in the tumor. Retroviral LacZ gene-containing vectors that were either pseudotyped for the gibbon ape leukemia virus (GALV-1) receptor or had specificity for the amphotropic murine leukemia virus (MLV-A) receptor were used for transduction of five SCLC cell lines differing by a range of MDR mechanisms. Transduction efficiencies in these cell lines were compared by calculating the percentage of blue colonies after X-Gal staining of the cells grown in soft agar. All examined SCLC cell lines were transducible with either vector. Transduction efficiencies varied from 5.7% to 33.5% independent of the presence of MDR. These results indicate that MDR does not severely impair transduction of SCLC cells, and that MLV-A as well as GALV-1 retroviral vectors are suitable for further development of gene therapy in SCLC.     

Dose-dependent transduction of vesicular stomatitis virus G protein-pseudotyped retrovirus vector into human solid tumor cell lines and murine fibroblasts.

We examined the transduction efficiency of a VSV-G (vesicular stomatitis virus G protein)-pseudotyped vector encoding beta-galactosidase (lacZ) into human solid tumor cell lines and murine fibroblasts, compared with that of an amphotropic vector carrying the same RNA sequence. The ratio of cells transduced with the VSV-G-pseudotyped vector corresponded closely to 1 - e(-m.o.i.), as predicted from a Poisson distribution of transduction to the entire cellular population, while this was not the case for the amphotropic vector. Here m.o.i. (multiplicity of infection) is defined as the ratio of input infectious units (titrated on the corresponding cell line) to the number of cells used for the transduction. At high m.o.i.s (values greater than 3), the VSV-G-pseudotyped vector transduced approximately 95% of the culture population of all cell lines examined. The transduction efficiency of the amphotropic vector, however, was not dose-dependent and reached a plateau or even decreased, especially at high m.o.i.; this may be attributable at least in part to the presence of envelope protein and noninfectious particles that compete for the receptor of infectious amphotropic virus. The copy number of integrated vector proviral DNA and the expression level of lacZ increased almost linearly with the dose of the VSV-G-pseudotyed vector, which could readily achieve multiple transduction of more than 10 copies per cell and afforded about 100-fold more transgene product than could be achieved with the amphotropic vector. These features of both the VSV-G-pseudotyped vector and the amphotropic vector were essentially unaffected by purification using centrifugation. These properties of the vector should be highly advantageous for gene transfer into entire populations of human tumor cell lines at a designed dosage.     

转IL—2基因的骨髓基质细胞对骨髓移植后免疫功能重建的促进作用

目的探讨了用逆转录病毒载体转入小鼠IL-2基因的基质细胞系QXMSC1对异基因骨髓移植后免疫功能重建的促进作用.方法将小鼠IL-2cDNA片段连接到逆转录病毒载体PLXSN上,构建重组逆转录病毒载体PL2SN(含小鼠IL-2cDNA).用磷酸钙共沉淀法将PL2SN转入单嗜性包装细胞系CRE,获得G418抵抗细胞株后,以上清感染双嗜性包装细胞系CRIP,G418筛选后获得高滴度的包装细胞系CRIPIL-2.感染NIH3T3细胞测定CRIPIL-2培养上清病毒滴度为3.4×105cfu/ml.感染骨髓基质细胞系QXMSC1(H-2d),G418筛选,有限稀释法得10个单克隆细胞株,选择表达IL-2最高的细胞株为实验用细胞QXMSC1IL-2,用于以后的实验.供体小鼠BALB/c(H-2d)骨髓以抗T细胞单抗anti-Thy1.2加补体去除骨髓中T细胞,受体小鼠C57BL/6(H-2b)经γ射线致死剂量照射后,输入供体骨髓1×107/只,同时输入基质细胞QXMSC1IL-25×105/只.在第30天、60天检测BMT小鼠脾细胞对LPS、ConA的反应,脾细胞产生IL-2的能力,BMT小鼠产生抗体生成细胞(PFC)的能力及产生DTH的能力.流式细胞仪检测了QXMSC1IL-2对BMT小鼠T细胞亚群的影响.结果电泳及酶切鉴定证实构建的PL2SN质粒.QXMSC1IL-2细胞系IL-2的分泌量为857U(1×106*24h).异基因骨髓移植,共输入QXMSC1IL-2能明显增加BMT小鼠脾细胞对LPS、ConA的反应.脾细胞产生IL-2的能力增强.PFC数目增加,DTH反应增强.T细胞亚群中CD4+/CD8+的比例有所恢复.结论转入IL-2的骨髓基质细胞系可促进骨髓移植后免疫功能重建.     

T cell suicide gene therapy to aid haematopoietic stem cell transplantation

Graft versus host disease (GVHD) is a T cell mediated phenomenon that arises following allogeneic haematopoietic stem cell transplantation, and may be particularly severe in the context of human leukocyte antigen (HLA) mismatched procedures. Although GVHD can be largely abrogated through T cell depletion, such measures result in loss of graft potency and reduced anti-viral and anti-leukaemic effects. The genetic modification of T cells to carry a suicide gene mechanism has been advocated as means of allowing T cells to be harnessed for their beneficial effects, and safely eliminated in the event of significant GVHD. The feasibility of the strategy has been demonstrated in clinical studies using T cells modified by retroviral transduction to encode the herpes simplex thymidine kinase (HSVTK) gene to treat patients with haematological malignancies. However, a number of limitations associated with current protocols have become apparent. Most notably, the process of retroviral transduction, which requires pre-activation of T cells, appears to impair subsequent functional potential. Efforts are now directed towards circumventing the pre-activation requirements of retroviral vectors by using alternative lentiviral systems, in association with improved suicide gene/prodrug combinations.     

Magnetic concentration of a retroviral vector using magnetite cationic liposomes

For tissue engineering purposes, retroviral vectors represent an efficient method of delivering exogenous genes such as growth factors to injured tissues because gene-transduced cells can produce stable and constant levels of the gene product. However, retroviral vector technology suffers from low yields. In the present study, we used magnetite nanoparticles and magnetic force to concentrate the retroviral vectors to enhance the transduction efficiency and to enable their magnetic manipulation. Magnetite nanoparticles modified with cationic liposomes were added to a solution containing a retroviral vector pseudotyped with vesicular stomatitis virus glycoprotein. The magnetic particles that captured the viral vectors were collected using a magnetic force and seeded into mouse neuroblastoma Neuro2a cells. The viral titer was up to 55 times greater (up to 3 x 10(8) infectious units/mL). Additionally, the magnetically labeled retroviral vectors can be directed to the desired regions for infection by applying magnetic fields, and micro-patterns of gene-transduced cell regions could be created on a cellular monolayer using micro-patterned magnetic concentrators. These results suggest that this technique provides a promising approach to capturing and concentrating viral vectors, thus achieving high transduction efficiency and the ability to deliver genes to a specific injured site by applying a magnetic field.