Efficient gene transfer into differentiated human trophoblast cells with adenovirus vector containing RGD motif in the fiber protein

Previously we reported fiber-modified adenovirus (Ad) vectors containing the Arg-Gly-Asp (RGD) motif on the HI loop of the fiber knob (Ad-RGD vectors) have high gene transfer efficacy into some human trophoblast cell lines. In the current study, we investigate transgene activity of Ad-RGD during differentiation of human cytotrophoblast BeWo cells into syncytiotrophoblast-like cells. Although cellular differentiation into syncytiotrophoblast cells was followed by a decrease in the coxsackievirus and adenovirus receptor levels on the cell membrane, the alphaVbeta3 and alphaVbeta5 integrin levels did not change. Conventional adenovirus vector had lower transduction activity in the differentiated cells than non-differentiated cells. In contrast, Ad-RGD vector had no influence on differentiation and had a ca. 2-5 fold higher transduction activity than that of the conventional Ad vector. Thus, Ad-RGD vector can be a powerful tool for gene transfer experiments in syncytiotrophoblast cells.     

Basic study for next-generation gene therapy products

Successful gene therapy depends largely on vectors that can efficiently deliver the therapeutic genes into the target tissues and cells. Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy because they can easily be grown to high titers and can efficiently transfer genes into both dividing and nondividing cells. However, there are some limitations such as the time-consuming and labor-intensive procedures for vector construction, coxsackievirus-adenovirus receptor (CAR)-dependent gene transfer, immunologic side effects, lack of tissue specificity, lack of regulation of gene expression, etc. In this paper, I review our approach to the development of advanced recombinant Ad vectors. The next generation of Ad vectors have not only become promising vectors for gene therapy but also important tools for gene transfer into mammalian cells.     

Approaches for generating recombinant adenovirus vectors.

Various methods have been developed to facilitate the generation of recombinant adenovirus vectors, and three commercially available methods have been most widely used: the homologous recombination method in E1-complement cell lines, the homologous recombination method in bacteria, and an in vitro ligation method based on simple routine plasmid construction. These methods can insert foreign genes not only into the E1 deletion region, but also into the E3 deletion region, thereby permitting the construction of a binary transgene expression system in which heterologous genes can be inserted into both the E1 and E3 regions. By modifying the latter two methods, fiber-mutant adenovirus vectors can be also constructed in order to modify vector tropism. In this paper, we review recent advances in the construction of first generation adenovirus vectors and fiber-modified adenovirus vectors.     

Transplacental drug delivery: gene and virus delivery to the trophoblast

The development of successful strategies for delivering genes to the placenta may provide new opportunities for modifying trophoblast function in order to learn more about trophoblast physiology and to offer novel therapeutic options for complications of pregnancy that result from placental dysfunction. Replication-deficient recombinant viral vectors are useful vehicles for introducing genes into cells in vitro and in vivo. Recombinant adenovirus and herpes simplex virus vectors are unable to efficiently infect and transduce terminally differentiated trophoblastic cells. However, recombinant adeno-associated virus vectors transduce terminally differentiated trophoblastic cells, and a Sindbis virus construct efficiently transduces and destroys trophoblastic cancer cells. We describe the features that make particular viral vectors attractive for gene transfer to trophoblastic cells.     

整合素靶向性病毒载体对肿瘤细胞基因转导的促进作用

在进行基因导入研究及临床试验中，目前采用的载体通常包括了病毒及非病毒两大类。对于非病毒载体用于基因转染有许多报道。而腺病毒作为基因载体，因其具有的高效转导能力及对分裂或非分裂细胞均能转导等优点而被广泛应用于针对疾病的基因治疗中。对腺病毒进入细胞机制进行的深入研究结果发现，腺病毒与细胞结合的第一步是与细胞表面的柯萨奇腺病毒受体（coxsackie adenovirus receptor，CAR）结合，第二步是利用其在五角体基（pentonbase）中的RGD序列和细胞表面的整合素结合，     

Anti-tumor responses induced by chemokine CCL19 transfected into an ovarian carcinoma model via fiber-mutant adenovirus vector

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an E1 cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-encoding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in B6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.     

Adenovirus vector-mediated gene transfer into stem cells

Stem cells, including embryonic stem (ES) cells, mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs), are defined by their capacity for self-renewal and multilineage differentiation. Efficient gene transfer into stem cells is essential for the basic research in developmental biology and for therapeutic applications in gene-modified regenerative medicine. Adenovirus (Ad) vectors, based on Ad type 5, can efficiently and transiently introduce the exogenous gene into many cell types via the primary receptor, coxsackievirus, and adenovirus receptor (CAR). However, some kinds of stem cells, such as MSCs and HSCs, cannot be efficiently transduced with conventional Ad vectors based on Ad serotype 5 (Ad5), because of the lack of CAR expression. To overcome this problem, fiber-modified Ad vectors and an Ad vector based on another serotype of Ad have been developed. Here, we review the advances in the development of Ad vectors suitable for stem cells and discuss their application in basic biology and clinical medicine.     

Tetracycline-regulatable adenovirus vectors: pharmacologic properties and clinical potential.

Stringent control of gene expression in human gene therapy strategies is important for both therapeutic and safety reasons. Replication-defective vectors derived from adenoviruses have been shown to be capable of highly efficient in vivo gene delivery to a wide variety of dividing and nondividing human cells. Here, we review the progress in the development of regulatable adenovirus vectors that allow gene expression to be tightly controlled by low concentrations of tetracyclines. As an example of the potential clinical utility of this technology, we highlight our results obtained in an immunotherapy model for prostate cancer with a tetracycline-regulatable adenovirus vector expressing the cytokine interleukin-12. Recombinant adenovirus vectors with tetracycline-regulatable gene expression provide new opportunities and improved safety for gene therapy applications in humans.     

Approaches and methods in gene therapy for kidney disease

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed and employed using nonviral, viral, and cellular vectors. The most efficient vector for in vivo transfection appears to be adenovirus. Glomeruli, blood vessels, interstitial cells, and pyelum can be transfected with high efficiency. In addition, electroporation and microbubbles with ultrasound, both being enhanced naked plasmid techniques, offer good opportunities. Trapping of mesangial cells into the glomeruli as well as natural targeting of monocytes or macrophages to inflamed kidneys are elegant methods for site-specific delivery of genes. For gene therapy in kidney transplantation, hemagglutinating virus of Japan liposomes are efficient vectors for tubular transfection, whereas enhanced naked plasmid techniques are suitable for glomerular transfection. However, adenovirus offers the best opportunities in a renal transplantation setup because varying parameters of graft perfusion allows targeting of different cell types. In renal grafts, lymphocytes can be used for selective targeting to sites of inflammation. In conclusion, for both in vivo and ex vivo renal transfection, enhanced naked plasmids and adenovirus offer the best perspectives for effective clinical application. Moreover, the development of safer, nonimmunogenic vectors and the large-scale production could make clinical renal gene therapy a realistic possibility for the near future.     

大鼠糖皮质激素受体基因腺病毒载体的构建及在肾系膜细胞中的表达

目的 利用AdEasy系统构建大鼠糖皮质激素受体(GR)基因重组腺病毒载体,转染大鼠肾小球系膜细胞(GMC),并通过Western Blot法检测GR蛋白的表达.方法 将质粒pcDNA1 Neo-Rat GR扩增、凝胶回收获得的Rat GR cDNA双酶切片段,插入腺病毒穿梭载体质粒pShuttle-CMV的巨细胞病毒(CMV)启动子下游,构建重组穿梭载体pShuttle-CMV-Rat GR,线性化后与骨架载体AdEasy-1在细菌BJ5183内经同源重组得到腺病毒重组质粒pAd-Rat GR,经人胚肾293A细胞包装后得到复制缺陷型重组腺病毒Ad-Rat GR;用包装后的病毒上清转染大鼠GMC,提取细胞中总蛋白,通过Western Blot方法 检测GR蛋白的表达.结果 连接、重组后通过酶切和测序法筛选出pAd-Rat GR重组质粒,经人胚肾293细胞包装,48 h后观察到增强型绿色荧光蛋白(EGFP)明显表达,氯化铯梯度离心纯化最终获得1×109 PFU /ml 滴度的重组病毒;用该滴度的Ad-Rat GR,转染大鼠GMC 48 h后提取细胞总蛋白,Western Blot检测GR蛋白有明显表达.结论 构建的大鼠GR基因腺病毒载体能够在大鼠GMC 中表达GR蛋白,并有上调作用.     

重组pAd/CMV/V5-DEST-p16载体的构建及其对人肝癌细胞的抑制作用

目的构建pAd/CMV/V5-DEST-p16重组腺病毒载体,并观察野生型p16基因对人肝癌细胞SMMC7721株的抑制作用。方法合成人p16-INK4表达基因。构建pENTR 1A-p16质粒。通过LR反应,入口克隆pENTR 1A-p16质粒的外源性合成的修饰后的p16 cDNA,取代目的载体pAd/CMV/V5-DEST中的ccdB基因,形成表达克隆pAd/CMV/V5-DEST-p16。测序证实质粒含有目的基因。PacI酶切后的重组腺病毒载体,通过脂质体2000介导,转染293A细胞,产生缺陷性的重组腺病毒。W estern blot分析显示在由重组腺病毒介导的野生型p16基因在肝癌细胞SMMC7721中能够表达蛋白。被感染的细胞的生长受抑制。结果构建了重组p16腺病毒载体,产生缺陷性的重组腺病毒,野生型p16基因对人肝癌细胞SMMC7721株有抑制作用。结论用通路克隆系统构建重组p16腺病毒载体稳定、可靠、方便,腺病毒能够介导野生型p16基因在肝癌细胞中表达,并抑制细胞的生长。     

Recombinant adeno-associated virus vectors efficiently transduce foreign gene into bovine aortic endothelial cells: comparison with adenovirus vectors

Because the features and kinetics of adeno-associated virus (AAV)-mediated gene transfer to endothelial cells (EC) are yet to be ultimately determined, we tested variables pertinent to the efficiency of AAV-mediated gene transfer to bovine aortic endothelial cells (BAEC). The variables with AAV vectors were compared with the better characterized adenovirus (Ad) vectors. There is a dose-response relationship between multiplicity of infection (moi) of AAV or Ad vectors and transduction efficiency in BAEC. The higher moi of AAV vectors achieved more than 80% of transduction efficiency in cultured BAEC. AAV and Ad vectors showed an incubation-time-dependent increase in transduction efficiency of LacZ gene to the BAEC up to 12 h of vector exposure. Although the similar kinetics of transduction efficiency of LacZ gene to BAEC was found in both vectors, the duration of gene expression was longer in AAV vector than that in Ad vectors in vitro. These results indicate that AAV-vector is efficient for gene transfer to EC, and higher moi of vectors or a longer period exposure of vectors to EC can facilitate efficient transduction of a foreign gene into cultured EC. For the duration of gene expression, the AAV vectors may be better than Ad vectors.     

Selectively replicating adenoviruses for cancer therapy: an update on clinical development

Adenoviruses can be engineered to replicate selectively in tumor cells but inefficiently in normal cells. ONYX-015 (CI-1042, dl1520; Onyx Pharmaceuticals Inc), which replicates selectively in cells deficient in the p53 pathway, was the first such adenovirus to reach clinical testing. Multiple trials of ONYX-015 in over 300 cancer patients, and trials with other selectively replicating adenoviruses, have established the safety of this approach. Evidence of anticancer activity in patients is encouraging. Recently, the first clinical trial of a selectively replicating adenovirus carrying an inserted transgene was reported. Adenoviruses with improved efficiency of replication, technologies for use of the viruses as vectors for anticancer gene therapy, and various other approaches, provide promising directions to develop selectively replicating adenoviruses into systemic therapy for metastatic cancer.     

Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro

AIM: To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth. METHODS: A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine. RESULTS: Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine. CONCLUSION: The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.     

siRNA腺病毒载体沉默MEKK3基因促进TRAIL诱导肝癌HCC-9204细胞凋亡

目的研究抑制丝裂原活化蛋白激酶/细胞外信号调节激酶-激酶3（MEKK3）基因表达促进TRAIL诱导人肝癌HCC-9204细胞凋亡的作用。方法构建人MEKK3基因的siRNA重组腺病毒载体,转导人肝癌HCC-9204细胞,以RTPCR和印迹法检测MEKK3 mRNA和蛋白的表达,筛选并建立稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株,以流式细胞仪检测TRAIL诱导细胞凋亡的情况。结果①成功构建并鉴定表达人MEKK3基因的siRNA重组腺病毒载体。②建立MEKK3基因有效且稳定沉默的人肝癌HCC-9204细胞株。③500 g/LTRAIL处理MEKK3基因沉默的HCC-9204细胞株,细胞凋亡率较野生HCC-9204细胞株显著提高。结论本研究成功建立有效且稳定沉默MEKK3基因表达的人肝癌HCC-9204细胞株模型,初步证实MEKK3基因表达与TRAIL诱导肝癌细胞凋亡的敏感性相关。     

Adenovirus-vectored vaccines

Background: Engineered adenoviruses are being increasingly explored as immunoprophylactic or immunotherapeutic vaccine vectors. Encouraging data from preclinical studies using human adenovirus vectors carrying different antigen genes have resulted in many currently ongoing clinical trials. Objective: The article seeks to review the current status of the use of adenoviruses as vaccine vectors. Methods: This review is based on the patent literature since 2000 pertaining to the development of adenovirus vaccine vectors for infectious and non-infectious diseases. Conclusion: Human adenovirus-vectored vaccines have important limitations that stem from their immunogenicity and restrict their utility. This has spurred intensive efforts to find alternative adenovirus vectors and strategies, each with its own advantages and shortcomings.     

Exploring DNA damage responses in human cells with recombinant adenoviral vectors

Recombinant adenoviral vectors provide efficient means for gene transduction in mammalian cells in vitro and in vivo. We are currently using these vectors to transduce DNA repair genes into repair deficient cells, derived from xeroderma pigmentosum (XP) patients. XP is an autosomal syndrome characterized by a high frequency of skin tumors, especially in areas exposed to sunlight, and, occasionally, developmental and neurological abnormalities. XP cells are deficient in nucleotide excision repair (affecting one of the seven known XP genes, xpa to xpg) or in DNA replication of DNA lesions (affecting DNA polymerase eta, xpv). The adenovirus approach allows the investigation of different consequences of DNA lesions in cell genomes. Adenoviral vectors carrying several xp and photolyases genes have been constructed and successfully tested in cell culture systems and in vivo directly in the skin of knockout model mice. This review summarizes these recent data and proposes the use of recombinant adenoviruses as tools to investigate the mechanisms that provide protection against DNA damage in human cells, as well as to better understand the higher predisposition of XP patients to cancer.     

Innate immune response induced by gene delivery vectors

Gene therapy is a clinical strategy that has the potential to treat an array of genetic and nongenetic diseases. Vectors for gene transfer are the essential tools of gene therapy. For gene therapy to be successful, an appropriate amount of the therapeutic gene must be delivered into the target cells without substantial toxicity. A major limitation of the use of gene therapy vectors is the innate immune responses triggered by systemic administration of such vectors. It is essential to overcome vector-mediated innate immune responses, such as production of inflammatory cytokines, the maturation of antigen-presenting cells and tissue damage, because the induction of these responses not only shortens the period of gene expression but also leads to serious side effects. Viral vectors (for example, adenovirus (Ad) vectors) have been assumed to be more potent in inducing innate immune responses in spite of their high transduction efficiency since they contain pathogenic proteins. However, recent studies have demonstrated that not only viral vectors but also nonviral vectors, such as lipoplex (liposome/plasmid DNA complex), can induce innate immune responses. Indeed, nonviral vectors including lipoplex induce comparable or larger levels of innate immune response than viral vectors. In this review, we present an overview of the innate immune responses induced by Ad vector and lipoplex, which are used primarily for in vivo gene transfer.     

Efficient gene transfer into human trophoblast cells with adenovirus vector containing chimeric type 5 and 35 fiber protein

Recombinant adenovirus (Ad) vectors based on Ad type 5 have been widely used for gene transfer experiments. Conventional Ad type 5 vectors have a narrow range of tropism and are limited by the size of the transgene that can be packaged. To overcome these limitations, we previously developed an Ad vector (Ad5/35 vector) containing a chimeric Ad type 5 and 35 fiber protein. In the current study, we evaluated the ability of the Ad5/35 vector to transfer genes into human trophoblast cell lines (JAR, JEG-3 and BeWo cells), which are used as in vitro models of human placenta. We compared the gene transfer efficiency of Ad5/35 to that of conventional Ad vector. We found that expression of CD46, which are receptors for Ad5/35 vector, are higher than that of coxsackievirus and adenovirus receptor in all 3 trophoblast cell lines, as determined by flow cytometry. Next, we compared the transducing activity of Ad5 vector and Ad5/35 vector that each expressed luciferase as a reporter gene. Ad5/35 vector had greater gene transfer activity than the conventional Ad vector in all 3 trophoblast cell lines (1.82-fold in JAR cells, 5.37-fold in BeWo cells, 6.11-fold in JEG-3 cells). Thus, Ad vector that contains chimeric type 5 and 35 fiber protein can be a powerful tool for gene transfer experiments in human trophoblast cell lines.