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1.
Rescue and production of vaccine and therapeutic adenovirus vectors expressing inhibitory transgenes
Gall JG Lizonova A EttyReddy D McVey D Zuber M Kovesdi I Aughtman B King CR Brough DE 《Molecular biotechnology》2007,35(3):263-273
Expression of certain transgenes from an adenovirus vector can be deleterious to its own replication. This can result in the
inhibition of virus rescue, reduced viral yields, or, in the worst case, make it impossible to construct a vector expressing
the inhibiting transgene product. A gene regulation system based on the tet operon was used to allow the rescue and efficient growth of adenovectors that express transgenes to high levels. A key advantage
to this system is that repression of transgene expression is mediated by the packaging cell line, thus, expression of regulatory
products from the adenovector are not required. This provides a simple, broadly applicable system wherein transgene repression
is constitutive during vector rescue and growth and there is no effect on adenovector-mediated expression of gene products
in transduced cells. Several high-level expression vectors based on first- and second-generation adenovectors were rescued
and produced to high titer that otherwise could not be grown. Yields of adenovectors expressing inhibitory transgene products
were increased, and the overgrowth of cultures by adenovectors with nonfunctional expression cassettes was prevented. The
gene regulation system is a significant advancement for the development of adenovirus vectors for vaccine and other gene transfer
applications. 相似文献
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The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life
cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently
and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We
also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments
that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology. 相似文献
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Designing gene delivery vectors for cardiovascular gene therapy 总被引:3,自引:0,他引:3
Baker AH 《Progress in biophysics and molecular biology》2004,84(2-3):279-299
Genetic therapy in the cardiovascular system has been proposed for a variety of diseases ranging from prevention of vein graft failure to hypertension. Such diversity in pathogenesis requires the delivery of therapeutic genes to diverse cell types in vivo for varying lengths of time if efficient clinical therapies are to be developed. Data from extensive preclinical studies have been compiled and a certain areas have seen translation into large-scale clinical trials, with some encouraging reports. It is clear that progress within a number of disease areas is limited by a lack of suitable gene delivery vector systems through which to deliver therapeutic genes to the target site in an efficient, non-toxic manner. In general, currently available systems, including non-viral systems and viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV), have a propensity to transduce non-vascular tissue with greater ease than vascular cells thereby limiting their application in cardiovascular disease. This problem has led to the development and testing of improved vector systems for cardiovascular gene delivery. Traditional viral and non-viral systems are being engineered to increase their efficiency of vascular cell transduction and diminish their affinity for other cell types through manipulation of vector:cell binding and the use of cell-selective promoters. It is envisaged that future use of such technology will substantially increase the efficacy of cardiovascular gene therapy. 相似文献
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Production of adenovirus vector for gene therapy 总被引:20,自引:0,他引:20
The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed. 相似文献
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The delivery of genetic material to mammalian cells is of great importance for modern fundamental biology, biomedicine, biotechnology, agriculture, and veterinary medicine. The development of new efficient techniques of gene transfer to human cells led to the advent of gene therapy, a novel approach to treating severe metabolic disorders, some viral infections (including HIV infection), autoimmune diseases, and genetic defects causing cancer. The review considers the main principles of constructing gene transfer and expression systems based on lentiviruses, a powerful tool for human gene therapy and transgenic research, with a special focus on the genome structure and life cycle of lentiviruses and the design and safety of lentiviral vector systems. 相似文献
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基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制. 相似文献
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Goutam Ghosh-Choudhury Yousef Haj-Ahmad Pamela Brinkley John Rudy Frank L. Graham 《Gene》1986,50(1-3):161-171
By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example. 相似文献
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目的采用体外连接技术构建含红色荧光蛋白(RFP)报告基因的重组腺病毒载体,为基因治疗与基因疫苗研究提供重要工具。方法将pTurbo RFP-N上的含红色荧光蛋白基因片段,经酶切连接定向克隆至转移载体pShuttle上,构成重组质粒pShuttle-TurboRFP-N。用I-CeuI/PI-SceI双酶切重组质粒pShuttle-TurboRFP-N和骨架质粒pH5′040 pkGFP-II,回收目的片段,连接,获得重组腺病毒载体pH5.′040.CMV.RFP-N。将其线性化后,经脂质体介导转染至AD293细胞内包装出重组腺病毒颗粒。通过荧光显微镜观察其在AD293细胞内的包装效率和RFP表达情况。扩增并再次感染AD293细胞检测病毒颗粒感染能力,并测定其生物活性及滴度。结果经酶切鉴定,证明已正确构建重组腺病毒载体AdH5.′040.CMV.RFP-N;经AD293细胞包装成具有感染性的病毒颗粒,并能在真核细胞中高效表达目的基因;扩增纯化后获得重组腺病毒颗粒数为3.6×1012vp/mL,滴度达1×1010pfu/mL。结论成功构建了携带红色荧光蛋白报告基因的重组腺病毒载体,并能够在AD293细胞中高效地表达,为基因治疗与基因疫苗研究提供重要工具。 相似文献
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Herpes simplex virus vectors for gene therapy 总被引:2,自引:0,他引:2
David S. Latchman 《Molecular biotechnology》1994,2(2):179-195
Herpes simplex virus (HSV) has a number of advantages as a vector for delivering specific genes to the nervous system. These
include its large size, wide host range, and its ability to establish long-lived asymptomatic infections in neuronal cells
in which a specific region of the viral genome continues to be expressed. Unfortunately, the large size of this virus and
difficulty in manipulating it has led to its use as a vector lagging behind that of other, smaller viruses such as the retroviruses.
In addition, the virus's ability to replicate lytically in the brain, under some circumstances, causing encephalitis, has
led to fears about its potential safety for ultimate use in humans. This review will discuss a number of new approaches that
are aimed at rendering simpler the insertion of foreign genes into the virus and making it as safe as possible. Ultimately,
these advances offer real hope for the use of HSV vectors in gene therapy procedures. 相似文献
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New adenovirus vectors for protein production and gene transfer 总被引:9,自引:0,他引:9
Bernard Massie Dick D. Mosser Maria Koutroumanis Isabelle Vitté-mony Linda Lamoureux France Couture Luc Paquet Claire Guilbault Julie Dionne Dounia Chahla Pierre Jolicoeur Yves Langelier 《Cytotechnology》1998,28(1-3):53-64
Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression
of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or
blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional
characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With
our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are
either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that
are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production
can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human
cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines.
Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about
6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus
vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated
in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible
at much lower levels of expression.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
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Ugai H Murata T Nagamura Y Ugawa Y Suzuki E Nakata H Kujime Y Inamoto S Hirose M Inabe K Terashima M Yamasaki T Liu B Nakade K Pan J Kimura M Saito I Hamada H Obata Y Yokoyama KK 《The journal of gene medicine》2005,7(9):1148-1157
BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world. 相似文献
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Development of methods to engineer gamma-retroviral vectors capable of transducing target cells in a cell-specific manner could impact the future of the clinical application of gene therapy as well as the understanding of the biology of transfer gene vectors. Two molecular events are critical for controlling the entry of gamma-retroviral vectors to target cells: binding to cell-surface receptors and the subsequent fusion of viral vector membrane and cellular membrane. In this report, we evaluated a method to incorporate a membrane-bound antibody and a fusogenic molecule to provide binding and fusion functions respectively, into gamma-retroviral vectors for targeted gene delivery. An anti-CD20 antibody and a fusogenic protein derived from Sindbis virus glycoprotein could be efficiently co-displayed on the surface of viral vectors. Vectors bearing anti-CD20 antibody conferred their binding specificity to cells expressing CD20. Enhanced in vitro transduction towards CD20-expressing cells was observed for gamma-retroviral vectors displaying both an antibody and a fusogen. We found that the biological activity of the fusogen played an important role on the efficiency of such a targeting strategy and were able to engineer several mutant forms of the fusogen exhibiting elevated fusion function to improve the overall efficiency of targeted transduction. We devised an animal model to show that subcutaneous injection of such engineered vectors to the areas xenografted with target cells could achieve targeted gene delivery in vivo. Taken together, we demonstrated as proof-of-principle a flexible and modular two-molecule strategy for engineering targeting gamma-retroviral vectors. 相似文献
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Synthetic gene transfer vectors based on zwitterionic nanoliposome-DNA assemblies (nanolipoplexes), formed by the mediation of magnesium ions, were prepared by a scalable method without employing volatile solvents, high-shear force treatments or extrusion. The zwitterionic nanolipoplexes (NLP) were formulated with PC (phosphatidylcholine) and DPPC (a natural lung surfactant) incorporating different amounts of cholesterol (CHOL). The resulting structures were characterised in terms of their morphology, size and DNA content. In addition, the toxicity and transfection efficiency of the nanolipoplexes were evaluated in cultured Chinese hamster ovary-K1 (CHO-K1) cells. The effects of the multivalent cation Mg2+ on nanoliposome-DNA transfection potency were evaluated. Formulations containing 10% CHOL showed maximum transfection efficiency and the optimum amount of Mg2+ ions for transfection with minimum cytotoxicity was ca. 20 mM. The zwitterionic formulations showed significantly less cytotoxicity compared to a commercially available cationic liposome reagent or polyethylenimine (PEI) while they were superior in terms of gene transfer potency. The zwitterionic vectors formulated in this study avoid the use of toxic cationic lipids as well as toxic solvents and may have potential application in gene therapy. The new method will enable scale-up and manufacture of safe and efficacious transfection vehicles required for preclinical and clinical studies. Based on the advantages and superiority of the formulated nanolipoplexes, this method allows for the acceleration of nanolipoplex formulation, enabling the rapid development and evaluation of novel carrier systems for genes and other drugs. 相似文献
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