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Background
Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA - bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions. 相似文献3.
BACKGROUND: Plasmid DNA vectors offer the potential of safe gene therapy avoiding viral vector-mediated toxicity and immunogenicity. As plasmid DNA is bacterial in origin, presence of bacterial lipopolysaccharide (LPS) or unmethylated CpG dinucleotides may stimulate host innate immunity. METHODS: Primary cultures of mouse and rat dendritic cells were established and incubated with bacterial lipopolysaccharide; immunostimulatory CpG oligodeoxynucleotide; control GpC oligodeoxynucleotide; and a range of (pVR1012) plasmids encoding transgenes with increasing CpG content (wild-type and mutant human preproinsulin; non-mammalian eukaryotic eGFP reporter gene; and bacterial beta-galactosidase reporter gene). IL-12 secretion was assayed to determine in vitro plasmid immunogenicity. Local inflammatory response following intramuscular injection of these plasmids, with or without a non-ionic carrier SP1017, was characterised in vivo. RESULTS: Dose-responsive LPS and CpG stimulation of IL-12 secretion from dendritic cells was demonstrated. All plasmids induced significant IL-12 secretion in comparison to control unstimulated cells. The beta-galactosidase plasmid had highest CpG content and induced significantly higher IL-12 secretion than constructs containing a eukaryotic transgene. Injection of rat muscle with the beta-galactosidase construct induced greater inflammatory response than human preproinsulin constructs. This was further enhanced by SP1017. At 2 days post-injection, monocyte/macrophage injection site infiltration predominated with CD8-positive lymphocytes predominating at 7 days. There was no evidence of transgene expression in infiltrating immune cells. CONCLUSIONS: Dendritic cell immunostimulation may be employed as an in vitro bioassay of innate immune response to plasmid DNA vectors during evaluation for clinical gene therapy. 相似文献
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Jechlinger W Azimpour Tabrizi C Lubitz W Mayrhofer P 《Journal of molecular microbiology and biotechnology》2004,8(4):222-231
DNA as an active agent is among the most promising technologies for vaccination and therapy. However, plasmid backbone sequences needed for the production of pDNA in bacteria are dispensable, reduce the efficiency of the DNA agent and, most importantly, represent a biological safety risk. In this report we describe a novel technique where a site-specific recombination system based on the ParA resolvase was applied to a self-immobilizing plasmid system (SIP). In addition, this system was combined with the protein E-specific lysis technology to produce non-living bacterial carrier vehicles loaded with minicircle DNA. The in vivo recombination process completely divided an origin plasmid into a minicircle and a miniplasmid. The replicative miniplasmid containing the origin of replication and the antibiotic resistance gene was lost during the subsequently induced PhiX174 gene E-mediated lysis process, which results in bacterial ghosts. The minicircle DNA was retained in these empty bacterial cell envelopes during the lysis process via the specific interaction of a membrane anchored protein with the minicircle DNA. Using this novel platform technology, a DNA delivery vehicle--consisting of a safe bacterial carrier with known adjuvant properties and minicircle DNA with an optimized safety profile--can be produced in vivo in a continuous process. Furthermore, this study provides the basis for the development of an efficient in vitro minicircle purification process. 相似文献
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Herrera AM Rodríguez EG Hernández T Sández B Duarte CA 《Biochemical and biophysical research communications》2000,279(2):548-551
DNA immunization technology is based on the availability of adequate vectors for cloning and expression of heterologous immunoactive proteins in mammalian cells. We have developed a family of DNA plasmid vectors suitable to manipulate antigen expression and location. Their in vitro and in vivo functionality and application are also reported. The developed immune response, the aspects considered for vector design, and the possible independent manipulation of both blocks for the generation of bicistronic constructs, make of the pAEC family of plasmid vectors a source for DNA vaccine candidate's development for further evaluation in human clinical trials, and for potential use in the gene therapy approach. 相似文献
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【目的】尝试构建表达小干扰RNA(small interfering RNA,siRNA)的小环载体,并初步鉴定其对乙肝病毒(hepatitis B virus,HBV)复制及其基因表达的抑制作用。【方法】设计并合成靶向HBV S区的siRNA,将其克隆到小环载体pMC.BESPX-MCS2上,测序正确后将重组体pMC-H1-siHBS-U6转化入感受态E.coliZYCY10P3S2T,然后在培养基中加入L-阿拉伯糖,诱导其降解细菌骨架,获取只含有目的基因表达盒的小环RNA干扰载体pmc-H1-siHBS-U6。将小环RNA干扰载体与HBV真核表达质粒pHBV1.3共转染Huh-7细胞,分别在转染后1-7天,ELISA法检测Huh-7细胞上清中的HBsAg、HBeAg,并且通过Real-time RT-PCR法分析干扰RNA对HBV DNA及mRNA的抑制效果。【结果】成功构建了靶向HBV S基因的siRNA小环表达载体pmc-H1-siHBS-U6。该载体能显著抑制Huh-7细胞HBsAg和HBeAg分泌,并且其抑制效果能够维持2-3周时间。Real-time PCR证实HBV的DNA与mRNA水平分别降低了71%和80%,而对照siRNA及空载体则无此作用。【结论】成功构建了靶向HBV的小环RNA干扰载体,并且其能稳定、高效、特异地抑制HBV基因的表达与复制,该研究不仅对探索HBV的基因治疗提供了重要线索,而且为RNA干扰的应用提供了新的运载体系。 相似文献
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Vaysse L Gregory LG Harbottle RP Perouzel E Tolmachov O Coutelle C 《The journal of gene medicine》2006,8(6):754-763
BACKGROUND: To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle. METHODS: We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo. RESULTS: In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR. CONCLUSIONS: Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors. 相似文献
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Lee Y Park EJ Yu SS Kim DK Kim S 《Biochemical and biophysical research communications》2000,272(1):230-235
We have constructed an expression vector, pCK, that is able to drive high levels of gene expression in the skeletal muscles of mice. pCK contains not only the full length immediate-early (IE) promoter of human cytomegalovirus but also its entire 5' untranslated region upstream from the start codon of the IE gene. In addition, pCK contains the kanamycin resistance gene, but lacks nucleotide sequences unnecessary for its function as a gene delivery vector, allowing the plasmid size to be 3.7 kb. pCK produced significantly higher levels of vascular endothelial growth factor 165 both in vitro and in vivo than the control vector, the structure of which is similar to naked DNA vectors employed in previous gene therapy trials. pCK would not only significantly increase the therapeutic effects of naked DNA gene therapy but also dramatically cut down the costs for production and treatment. 相似文献
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Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI). 总被引:29,自引:13,他引:16
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P Renbaum D Abrahamove A Fainsod G G Wilson S Rottem A Razin 《Nucleic acids research》1990,18(5):1145-1152
We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases. 相似文献
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Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression
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Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.Efficient delivery and expression of foreign genes are of great importance in medicine and basic science. In many gene therapy applications, expression of the therapeutic gene would be required for the lifetime of the patient, yet many vector systems display only transient expression, lasting as little as a few days or weeks. Helper-dependent adenovirus (hdAd) vectors can enhance the duration of expression of a therapeutic gene; studies of mice and nonhuman primates have yielded several years of gene expression after a single administration (28). Indeed, several studies have described lifelong expression of a gene and persistent phenotypic correction in mouse models of human disease (18, 26, 42).Most hdAds contain noncoding “stuffer” DNA to maintain the size of the vector within appropriate limits for efficient DNA packaging; vectors constructed below ∼27 kb undergo DNA rearrangement in order to increase the size of the genome to 27 to 38 kb (31, 38). Interestingly, the nature of the stuffer DNA included in the hdAd has a significant effect on the function of the vector. An hdAd vector containing 22 kb of eukaryotic DNA (hdAd-euk) expressed a transgene to a higher level and for a longer duration than a vector containing 22 kb of prokaryotic DNA (hdAd-prok), both in vitro and in vivo (29). The genomes of the two vectors persisted at similar levels within the livers of transduced mice, suggesting that incorporation of prokaryote-derived stuffer DNA into an hdAd leads to the shutoff of associated transgenes. As a result of these observations, most current hdAd vectors are constructed using stuffer DNA derived from eukaryotic sources (27).Silencing of transgenes associated with prokaryotic DNA is not unique to hdAd. Removal of the bacterial origin of replication and antibiotic resistance gene from herpes simplex virus (HSV) amplicons resulted in a 20-fold improvement in gene expression in normal human fibroblasts in vitro, and more-persistent reporter gene expression in nude mice, compared to amplicons retaining the bacterial elements (39). Similarly, removal of bacterial sequences from plasmids results in significantly improved transgene expression in vitro and in vivo (2, 3, 34). For both plasmid and HSV amplicons, the mechanisms by which the bacterial sequences impair transgene expression are not fully understood. However, the bacterial sequences appear to nucleate the formation of a repressive chromatin structure(s) that spreads to the transgene (4, 39).In this study, we experimentally address the mechanism behind the repressive effects of prokaryotic DNA on gene expression in hdAd vectors. We found that prokaryotic DNA inhibits eukaryotic gene expression in cis, via induction of histone deacetylation, which is independent of DNA methylation. Furthermore, our data indicate that Sp100 and Daxx are involved in repressing the expression of genes associated with prokaryotic DNA. 相似文献
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Lina Li Emilios K. Dimitriadis Yu Yang Juan Li Zhenhua Yuan Chunping Qiao Cyriaque Beley Richard H. Smith Luis Garcia Robert M. Kotin 《PloS one》2013,8(8)
Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×109 Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA. 相似文献
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Thomas Skopek Deborah Marino Kristy Kort Judith Miller Myrna Trumbauer Shobhna Gopal Howard Chen 《Mutation research》1998,400(1-2):77-88
Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences. This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base. To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences. This gene was inserted into a lambda vector and used to construct trangenic mice that undergo vector rescue from genomic DNA upon in vitro packaging. Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue™ transgenic mice. Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10−5 range and were predominantly base pair substitutions, similar to results seen in Big Blue™. However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI. Unexpectedly, 23% of the spontaneous mrkII mutations were GC → AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII. Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue™. Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events. This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets. 相似文献
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Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders. 相似文献
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The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression 总被引:1,自引:0,他引:1
BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors. 相似文献