一种高效感染血液细胞的新型靶向性腺病毒载体的构建

人C组5型腺病毒(Ad5)载体能够有效感染上皮来源的细胞,但对造血细胞的感染效率很低,限制了其在造血调控基础研究以及血液病基因治疗中的应用。为了建立高效感染血液细胞的新型靶向性腺病毒载体系统,对5型腺病毒载体的纤维顶球进行了改造,以AdEasy系统为基础,应用递归PCR的方法人工合成人B组11p型腺病毒的部分纤维(fiber)基因,采用一系列分子生物学方法将其替换AdEasy骨架质粒中的人5型腺病毒的fiber基因,得到新的腺病毒骨架质粒命名为pAdEasy-1/F11p,应用带有GFP报告基因的穿梭质粒pShuttle-GFP与AdEasy-1/F11p腺病毒DNA在BJ5183细菌内重组得到重组腺病毒质粒,将其转染293细胞获得重组腺病毒,命名为Ad5F11p-GFP。以Ad5-GFP作对照,同时感染K562、U937等白血病细胞系,流式细胞仪检测GFP的表达。初步检测结果显示:在10MOI时,Ad5F11p-GFP能够有效感染K562、U937等白血病细胞系,感染细胞效率>90%,对照Ad5-GFP感染细胞效率<30%,这表明改建后的腺病毒AdEasy-1/F11p可以高效介导基因转移到血液细胞,是一种很好的血液细胞靶向性腺病毒载体。     

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells

BACKGROUND: A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)-based vector-mediated gene transfer into malignant hematopoietic cells. Fiber-retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells. METHODS: For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the PGK promoter with Ad35 fiber receptor specificity was generated (Ad5F35-GFP). The Ad5F35-GFP vector-mediated gene transfer efficiency was compared with a fiber non-modified Ad5-GFP vector, which also encodes the GFP gene under the control of the PGK promoter. RESULTS: We demonstrated that a variety of Ad5-refractory malignant myeloid and B lymphoid cell lines were highly permissive to the Ad5F35-GFP vector infection. Importantly, primary chronic myeloid leukemic (CML) cells and chronic lymphocytic leukemia (CLL) B cells were superiorly transduced by the Ad5F35-GFP vector at a multiplicity of infection (MOI) of 100 compared with the Ad5-GFP vector. CONCLUSIONS: Our study will facilitate the generation of fiber-retargeted adenoviral vectors and enable transient genetic manipulation of primary malignant hematopoietic cells.     

复制缺陷型腺病毒载体介导的人凝血因子Ⅷ基因在体内外的高效转移和表达

为了构建含人凝血因子Ⅷ基因的重组腺病毒载体系统,首先构建了含ＦⅧ（Ｂ结构域缺失型）的载体质粒ｐＡｄ－ｈＦⅧ和插入内含子结构的ｐＡｄＩ－ｈＦⅧ,经脂质体介导,证明两质粒的目的基因均能在ＣＯＳ－７细胞中表达有凝血活性的ＦⅧ因子,其中经纯化的载体质粒ｐＡｄ－ｈＦⅧ与ｐＢＨＧ１０经脂质体介导,共转染至２９３包装细胞,经同源重组和Ｅ１蛋白反式互补,形成Ｅ１／Ｅ３缺失型重组腺病毒载体颗粒Ａｄ－ｈＦⅧ．ＰＣＲ检则到病理２９３培养液上清的病毒ＤＮＡ中具有目的基因扩增片段,证明病毒ＤＮＡ含有ＦⅧ基因．经扩增、纯化、滴度测定,用于感染离体细胞ＣＯＳ－７、Ａ５４９、Ｌ９２９、２９３．证实该病毒能在上述细胞中高效转移和表达目的基因,其表达量具有一定范围内的剂量依赖性．ＣＯＳ－７细胞中,ＭＯＩ＞１０００时,有细胞毒效应,表达量反而下降．经耳缘静脉注射Ａｄ－ｈＦⅧ至家兔后,１～４周能测到一定水平的ＦⅧ蛋白,１周内升至最高峰．免疫组化研究表明感染的离体细胞和家兔肝等组织均有ＦⅧ表达,其中肝脏表达最高．为甲型血友病的基因治疗开辟了新途径     

Efficient adenoviral-mediated gene delivery into porcine mesenchymal stem cells

Mesenchymal stem cell (MSC) mediated gene therapy research has been conducted predominantly on rodents. Appropriate large animal models may provide additional safety and efficacy information prior to human clinical trials. The objectives of this study were: (a) to optimize adenoviral transduction efficiency of porcine bone marrow MSCs using a commercial polyamine-based transfection reagent (GeneJammer, Stratagene, La Jolla, CA), and (b) to determine whether transduced MSCs retain the ability to differentiate into mesodermal lineages. Porcine MSCs (pMSCs) were infected under varying conditions, with replication-defective adenoviral vectors carrying the GFP gene and GFP expression analyzed. Transduced cells were induced to differentiate in vitro into adipogenic, chondrogenic, and osteogenic lineages. We observed a 5.5-fold increase in the percentage of GFP-expressing pMSCs when adenovirus type 5 carrying the adenovirus type 35 fiber (Ad5F35eGFP) was used in conjunction with GeneJammer. Transduction of pMSCs at 10.3-13.8 MOI (1,500-2,000 vp/cell) in the presence of Gene Jammer yielded the highest percentage of GFP-expressing cells ( approximately 90%) without affecting cell viability. A similar positive effect was detected when pMSCs were infected with an Ad5eGFP vector. Presence of fetal bovine serum (FBS) during adenoviral transduction enhanced vector-encoded transgene expression in both GeneJammer-treated and control groups. pMSCs transduced with adenovirus vector in the presence of GeneJammer underwent lipogenic, chondrogenic, and osteogenic differentiation. Addition of GeneJammer during adenoviral infection of pMSCs can revert the poor transduction efficiency of pMSCs while retaining their pluripotent differentiation capacity. GeneJammer-enhanced transduction will facilitate the use of adenoviral vectors in MSC-mediated gene therapy models and therapies.     

Rapid titration of adenoviral infectivity by flow cytometry in batch culture of infected HEK293 cells

There is a constant and growing interest in exploitingadenoviruses as vectors for gene therapy when transientexpression of a therapeutic protein is necessary. Therequirement for an increased viral titre has prompted asearch for techniques by which this virus may be assayedwith greater speed and simplicity. Conventional plaqueassay for quantification of adenoviral vectors titre incurrent use is laborious and time-consuming (up to 14days). We report herein a method for the monitoring ofadenovirus expressing green fluorescent protein thatincorporates rapid and easy sample handling by means offlow cytometric analysis. Cells (HEK293) were infectedwith adenovirus at various multiplicity of infection(MOI), harvested 17 to 20 h post infection and analysedby flow cytometry. Assumptions were made that onefluorescent cell was infected by a single infectiousparticle at a relatively low MOI. The adenoviral titrewas subsequently estimated from cell analysis in arelatively short time. The results obtained with an E1-complementing cell line (HEK293) were compared with thatobtained using a non-complementing cell line (A549). APoisson distribution successfully modelled the profile ofinfection as a function of MOI. This provided a betterunderstanding of adenoviral infection at the earlieststage possible. Monitoring of GFP fluorescence and viruspropagation in a batch culture of infected cells wassubsequently used as a practical application of thevalidated method.     

Ad-IL-24对SGC-7901胃癌细胞生长抑制的体外实验

旨在研究携带人IL-24基因的腺病毒表达载体(Ad-IL-24)对SGC-7901人胃癌细胞的生长抑制作用并分析其分子机制。以不同MOI(感染复数)的Ad空载体腺病毒感染SGC-7901人胃癌细胞,筛选出最佳感染剂量;Ad-IL-24以最佳感染剂量感染SGC-7901胃癌细胞,RT-PCR法和Western blotting法检测腺病毒介导的IL-24基因在SGC-7901胃癌细胞中的转录;MTT法检测Ad-IL-24对SGC-7901胃癌细胞的生长抑制作用,流式细胞仪检测其诱导SGC-7901人胃癌细胞凋亡和细胞周期改变的效应,Hoechst33258染色荧光显微镜检测其诱导胃癌细胞凋亡的核形态改变;RT-PCR半定量法进一步检测SGC-7901胃癌细胞中凋亡相关基因的转录。结果显示,100MOI为感染SGC-7901胃癌细胞的腺病毒最佳感染剂量;Ad-IL-24能成功介导IL-24基因在SGC-7901胃癌细胞中转录性表达;Ad-IL-24感染SGC-7901胃癌细胞后,能明显抑制胃癌细胞生长和诱导凋亡;Ad-IL-24能显著上调SGC-7901胃癌细胞中bax、caspase-3和p53的表达和下调bc...     

E1/E3缺失型腺病毒载体引起细胞周期G_2/M阻滞

腺病毒载体广泛应用于基因治疗和转基因研究 ,目前常用的E1 E3缺失型复制缺陷腺病毒载体虽然失去了病毒复制必需的E1基因 ,但载体上的其它病毒基因仍能在宿主细胞内表达 .为研究这些基因对细胞的毒性作用 ,选择了 3种携带没有明显细胞毒性外源基因的腺病毒载体 ,观察感染 2种肿瘤细胞后细胞核形态改变 ,并用流式细胞仪检测细胞周期及凋亡情况 .发现大剂量重组缺陷型腺病毒感染细胞后引起细胞变圆 ,核增大 ,细胞周期阻滞于G2 M期 ,继而染色质凝聚 ,细胞发生坏死或凋亡 ;各种腺病毒载体造成G2 M阻滞所需感染量不同 ,但都随时间延长和感染量增加而加重 .这些结果提示腺病毒基因对细胞的影响是多方面的 ,在以此类病毒载体进行基因转移和基因治疗的研究中 ,精确滴定病毒滴度和转导效率非常重要 ,腺病毒基因表达造成的毒副作用给此类研究增加了变数     

腺病毒载体衣壳蛋白质的遗传修饰

腺病毒载体是最早用于基因治疗研究的病毒载体之一,也是目前肿瘤基因治疗中最为常见的病毒载体之一,其主要通过靶细胞表面的天然柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)感染宿主细胞。由于大多数肿瘤细胞表面该受体表达水平较低,降低了腺病毒载体对靶细胞的感染效率,从而制约了腺病毒载体在肿瘤基因治疗中的应用。因此,如何提高腺病毒载体对靶细胞的感染效率是腺病毒载体应用于肿瘤基因治疗的关键。目前对腺病毒载体衣壳蛋白质(capsid protein)的遗传修饰是提高其对宿主细胞感染效率的主要途径。本文将对这一领域的主要研究进展作一综述,为该方面的研究提供有用的信息。     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Trans-complementing adenoviral vectors for oncolytic therapy of malignant melanoma

BACKGROUND: Despite attempts to develop efficient viral-based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation. METHODS: We used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans-complementing replication-incompetent adenoviral vectors that resulted in tumor-restricted oncolysis. We combined an E1-deleted non-replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4-deleted/E4orf4-only expressing adenovirus, to allow full replication competence when tumor cells were co-infected with both vectors. RESULTS: A375 tumors showed apoptosis at the ultrastructural level after transduction with the trans-complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co-localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans-complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans-complementing system of viruses alone, and long-term survival was only seen in the trans-complementing vector treatment group. Intraperitoneal administration of a pseudo-wild-type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans-complementing vectors resulted in only mild liver abnormalities. CONCLUSIONS: The trans-complementing vector approach using a combination of E1- and E4-deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication-competent vectors by requiring the presence of both vectors in a cell to achieve replication competence.     

Fiber-knob modifications enhance adenoviral tropism and gene transfer in malignant glioma

BACKGROUND: Malignant gliomas remain refractory to Ad5-mediated gene therapy due to deficiency of the coxsackie adenovirus receptor on tumor cells. The purpose of this study was to evaluate whether changes in adenoviral tropism can enhance gene transfer in the context of malignant glioma. METHODS: We have identified several receptors that are over-expressed on tumor cells and created a series of pseudotyped Ad5 vectors that recognize these receptors: Ad5-RGD which binds alpha(v)beta3/alpha(v)beta5 integrins; Ad5/3 which contains adenovirus serotype 3 knob and binds to CD46; Ad5-Sigma which incorporates the reovirus sigma knob and binds to junctional adhesion molecule-1; and Ad5-pk7 which contains the polylysine motif and binds heparan sulfate proteoglycans. We also investigated the Ad5-CAV1 vector, which contains the knob of canine adenovirus type 1, a virus previously shown to infect glioma via an unknown mechanism. In this study, we compared these modified vectors for their ability to promote the expression of luciferase transgene both in vitro and in vivo. RESULTS: Our results indicate that all five modified vectors attained higher mean luciferase activity vs. control. Among them, Ad5-CAV1 and Ad5-pk7 attained the highest transduction efficiency independent of different tumor lines or infection time. Ad5-Sigma and Ad5-pk7 also demonstrated the least nonspecific infection in normal human astrocytes. Most importantly, Ad5-pk7 achieved 1000-fold increased transgene expression in human glioma xenografts in vivo. CONCLUSIONS: These results indicate that modifications of adenoviral tropism can enhance gene transfer in tumors that are poorly susceptible to adenoviral vectors and warrant further development of Ad5-pk7 for glioma gene therapy.     

经NGR肽段遗传修饰的携带三种报告基因的腺病毒载体的构建及其实验研究

NGR(Asn-Gly-Arg)是通过噬菌体展示技术筛选出来的能够和肿瘤新生血管特异结合的三肽模体,可以将多种药物分子和病毒载体靶向运输到肿瘤或者进行血管再生的组织中。为此构建了腺病毒衣壳蛋白knob的HI环(HI-loop)经NGR肽段修饰的并同时表达三种报告基因的腺病毒载体Ad5/E1-mCherry/E3-luciferase-2A-eGFP/knob-NGR。体内、外实验研究表明,该病毒载体可成功表达三种报告基因;经NGR肽段修饰的腺病毒载体对人乳腺癌细胞系MDA-MB-231的感染效率高于未经修饰的对照腺病毒Ad5CMVeGFP。该载体的成功构建为进一步研究经NGR肽段修饰的腺病毒在肿瘤动物模型体内的靶向性及经NGR肽段修饰的并携带治疗基因的实验治疗研究奠定了基础。     

Adenovirus vector production using low-multiplicity infection of 293 cells

The use of low-multiplicity infection of 293 cells in static culture with regular medium replacement was investigated for efficient large-scale production of adenovirus vectors for gene therapy applications. An adenovirus vector carrying the enhanced green fluorescent protein gene (Ad EGFP) was used to infect 293-F cells at a low multiplicity of infection (MOI) of 0.00001–0.1 transductional unit (TU) cell⁻¹. The cells, which have the ability to grow in suspension, were incubated in T-flasks and the serum-free culture medium was replaced with fresh medium via centrifugation every 2 days. Because only a small proportion of cells were initially infected at low MOIs (<1 TU cell⁻¹), uninfected cells continued to grow until they were infected by progeny adenoviruses released from previously infected cells. When 293-F cells at a relatively low density of 1 × 10⁵ cells cm⁻³ were infected with Ad EGFP at a low MOI of 0.001 TU cell⁻¹, the vector yield was 2.7-fold higher than the maximum yield obtained with high-multiplicity infection (MOI = 10 TU cell⁻¹) in batch culture. These results indicate that efficient adenovirus vector production using low MOIs is achieved by minimization of either nutrient depletion and/or accumulation of inhibitory metabolites in the culture medium.     

Cardiomyocyte-specific gene expression following recombinant adeno-associated viral vector transduction

Recombinant adeno-associated viral (rAAV) vectors hold promise for delivering genes for heart diseases, but cardiac-specific expression by the use of rAAV has not been demonstrated. To achieve this goal rAAV vectors were generated expressing marker or potentially therapeutic genes under the control of the cardiac muscle-specific alpha myosin heavy chain (MHC) gene promoter. The rAAV-MHC vectors expressed in primary cardiomyocytes with similar kinetics to rAAV-CMV; however, expression by the rAAV-MHC vectors was restricted to cardiomyocytes. rAAV vectors have low cytotoxicity, and it is demonstrated here that rAAV fails to induce apoptosis in cardiomyocytes compared with a recombinant adenoviral vector. rAAV-MHC or rAAV-CMV vectors were administered to mice to determine the specificity of expression in vivo. The rAAV-MHC vectors expressed specifically in cardiomyocytes, whereas the control rAAV-CMV vector expressed in heart, skeletal muscle, and brain. rAAV-MHC transduction resulted in long term (16 weeks) expression of human growth hormone following intracardiac, yet not intramuscular, injection. Finally, we defined the minimal MHC enhancer/promoter sequences required for specific and robust in vivo expression in the context of a rAAV vector. For the first time we describe a panel of rAAV vectors capable of long term cardiac specific expression of intracellular and secreted proteins.     

Reconstitution of ventricular myosin with atrial light chains 1 improves its functional properties

Atrial light chain 1 (ALC-1) is expressed in embryonic and hypertrophied human ventricles but not in normal adult human ventricles. We investigated the effects of recombinant human atrial light chains (hALC-1) on the structure and enzymatic activity of synthetic filaments of ventricular myosin. The endogenous ventricular myosin light chain 1 (VLC-1) was partially replaced by recombinant hALC-1 yielding hALC-1 levels of 12%, 24% and 42%. This reconstitution of ventricular myosin with hALC-1 did not change the length of synthetic myosin filaments but led to more rounded myosin heads in comparison with those of control filaments. Actin-activated ATPase activity of myosin, a parameter of functional activity of molecular motor, amounted to 79.5 nmol Pi/mg per min in control myosin filaments. Reconstitution with hALC-1 caused a profound increase of the actin-activated myosin ATPase activity in a dose dependent manner, for example, synthetic myosin filaments formed with 12%, 24% and 42% hALC-1 reconstituted myosin revealed the actin-activated ATPase activity increased by 18%, 26% and 36%, respectively, as compared to control. These results strongly suggest that in vivo expression of ALC-1 enhances ventricular myosin function, thereby contributing to cardiac compensation.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Cardiac-specific gene expression facilitated by an enhanced myosin light chain promoter

BACKGROUND: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. METHODS: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. RESULTS: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc). DISCUSSION: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.