The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Zhi-ying Chen - One of the best experts on this subject based on the ideXlab platform.
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calcium phosphate nanoneedle based gene delivery system for cancer genetic immunotherapy
Biomaterials, 2020Co-Authors: Cheng-yi He, Peifa Zhang, Xiaojuan Pang, Guochuang Chen, Ping Chen, Jing Zhao, Zhiyong Wang, Zhi-ying ChenAbstract:Abstract Ovarian cancer has become one of the most common gynecological cancers with a high mortality. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to active immune responses against cancer, the absence of efficient in vivo gene delivery technique is still an obstacle in clinical application. To overcome these problems, a Minicircle DNA vector encoding humanized anti-EpCAM/CD3 bispecific antibody (BsAbEPH) has been constructed. Moreover, different shapes of calcium phosphate (CaPO) biomaterials were prepared. Specifically, the CaPO-nanoneedle-mediated “cell perforation” transfection technology achieves high levels of gene expression in peritoneal cavity. In an intraperitoneal xenograft model with human ovarian cancer cell line SKOV3, the CaPO-nanoneedle/Minicircle DNA system expressed BsAbEPH resulted in significant retardation of cancer growth and extension of mouse life-span with limited toxicity. And this system can be made as off-the-shelf and easy-to-use products. Therefore, CaPO-nanoneedle based non-viral gene delivery technology will have great potential in clinical application.
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synthesis of amphiphilic poly β amino ester for efficiently Minicircle DNA delivery in vivo
ACS Applied Materials & Interfaces, 2016Co-Authors: Jing Zhao, Zhiyong Wang, Ping Huang, Yan Tan, Xiaohu Hou, Liping Zhang, Zhi-ying ChenAbstract:Minicircle DNA (mcDNA) is a kind of enhanced nonviral DNA vector with excellent profiles in biosafety and transgene expression. Herein, we reported a novel amphiphilic polymer comprising polyethylenimine(PEI) modified Poly(β-amino ester) PEI–PBAE(C16) for efficient mcDNA delivery in vivo. The synthesized polymer could condense mcDNA into nanoscaled structure and exhibited efficient gene transfection ability without detectable cytotoxicity. Importantly, when injected into mouse intraperitoneally, these PEI–PBAE(C16) nanocomplexes were able to result in high level of trangene expression which lasted at least 72 h. Overall, these results demonstrated the PEI–PBAE(C16) can mediate effective and safe gene delivery in vivo with clinical application potential.
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synthesis and characterization of low molecular weight polyethyleneimine terminated poly β amino ester for highly efficient gene delivery of Minicircle DNA
Joint International Conference on Information Sciences, 2016Co-Authors: Jing Zhao, Zhiyong Wang, Ping Huang, Yan Tan, Lei Yang, Hong Liu, Jiajia Pan, Zhi-ying ChenAbstract:Gene therapy has held great promise for treating specific acquired and inherited diseases. However, the lack of safe and efficient gene delivery systems remains as the major challenge. Poly(β-amino ester)s (PBAEs) have attracted much attention due to their outstanding properties in biosafety, DNA delivery efficiency and convenience in synthesis. In this paper, we reported the further enhancement of the PBAE functions by increasing its positive charge through conjugating with low molecular weight polyethylenimine (LPEI). The resulted LPEI-PBAE polymer was able to condense Minicircle DNA (mcDNA) forming nanoparticles with a diameter of 50-200nm. Furthermore, as compared to parental PBAE and a commercial transfection reagent very common in laboratory application, the LPEI-PBAE demonstrated significantly higher transfection efficiency with little cytotoxicity. These results suggested LPEI-PBAEs are worthy of further optimization for gene therapy applications.
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637 treatment of human b lymphoma by a Minicircle DNA vector expressing the cd3xcd20 bispecific antibody in a xenograft mouse model
Molecular Therapy, 2015Co-Authors: Jing Zhang, Cheng-yi He, Yujiang Zhong, Zhi-ying ChenAbstract:Breakthroughs have been achieved in cancer immunotherapies recently. Some patients experienced a durable remission or even cure by the treatments activating patients’ own T cells. However, these single target technologies have failed to result in any therapeutic effect in a large percent of patients, and evidence suggests that further advances depend on the effective strategy for coping with cancer heterogeneity and dynamics. We hypothesize that Minicircles, a class of optimized DNA vectors, are able to cope with these problems; that is to apply Minicircles to express a panel of engineered antibodies capable killing all cell lineages in a cancer by inducing antibody-dependent cellular cytotoxicity or retargeting CTLs to become anticancer T cells. To prove this concept, we constructed a Minicircle to express a bispecific antibody (BsAB) capable of binding CD3 on T cell and CD20 on B-lymphoma cell simultaneously, and tested if it could function like its BsAb counterpart blinotumomab in treating B-lymphoma. Six to eight week-old female SCID/Beige mice were inoculated with 2×5e5 Raji cells through tail vein injection, and 4 μg of the Minicircle was injected into the mouse liver via hydrodynamic technique 24 hours later. Selected mice received 3 intravenous doses of human DC-CIK cells, 5e6 each, every other day starting 2 days after Raji cell inoculation. All the 14 Raji cell-bearing mice receiving no further treatment died in 3 weeks, the 10 mice that received DC-CIDK as well died in 7 weeks, while the10 mice received both MC. CD3xCD20 and DC-CIK survived for additional 5 weeks. Histology examination demonstrated human CD3-positive T cells and CD20-positive B-lymphoma cells in spleens. In another fine-tuning experiment, we imaged the mice receiving the same number of Raji cell expressing luciferase gene when they demonstrated paralyzed rear legs, the typical syndrome indicating the late stage of this disease. We observed strong luciferase signal along the hips and spinal of the mice. Taken together, these data demonstrated that we have successfully established the mouse model of human B-lymphoma, and that minicrcle was able to generate therapeutic level of BsAb in vivo. Further experiments are undergoing to characterize the Minicircle-BsAb anti-cancer system. As compared to the virus vectors, Minicircle is safer, more convenient to make, easier to target multiple cancer cell lineages simultaneously, our efficacy data support the notion that Minicircles have the potential to serve as powerful immunotherapy vectors in fighting human cancer.
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increasing the Minicircle DNA purity using an enhanced triplex DNA technology to eliminate DNA contaminants
Molecular therapy. Methods & clinical development, 2015Co-Authors: Xiaohu H Hou, Xiaoyan Y Guo, Yusheng Chen, Zhi-ying ChenAbstract:DNA vectors for human gene therapy have to meet the efficacy and safety requirements. Minicircles (MCs), a class of optimized DNA vectors free of plasmid backbone (PB) DNAs, have emerged as promising candidates because of their superior transgene expression profiles. However, the existence of impure DNAs, including the unrecombined MC producing plasmid (PP) and PB circle, in the MC products made using the current technologies exceed the safety limit. Here, we report the development of an enhanced triplex DNA (TriD) technology to eliminate almost all the impure DNAs from the MC products. To do this, a pair of optimized TriD forming sequences was placed to flank the kanamycin resistance gene in the PP. The MC products were incubated with a biotinylated TriD forming DNA oligonucleotide (olig), and the resulted TriDs were removed by binding to streptovidin-coated magnetic beads. Consequently, the residual impure DNAs were 0.03% or less in the final MC products. The reproducibility of this technique was confirmed with MCs of various transgene expression cassettes, sizes, and quantities, suggesting its great potential in making high quality MC for human gene therapy.
Mark A. Kay - One of the best experts on this subject based on the ideXlab platform.
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a mini intronic plasmid mip a novel robust transgene expression vector in vivo and in vitro
Molecular Therapy, 2013Co-Authors: Feijie Zhang, Mark A. KayAbstract:The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the Minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with Minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.
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Minicircle DNA vectors achieve sustained expression reflected by active chromatin and transcriptional level
Molecular Therapy, 2013Co-Authors: Lia Gracey E Maniar, Zhi-ying Chen, Andrew Fire, Jay M Maniar, Mark A. KayAbstract:Current efforts in nonviral gene therapy are plagued by a pervasive difficulty in sustaining therapeutic levels of delivered transgenes. Minicircles (plasmid derivatives with the same expression cassette but lacking a bacterial backbone) show sustained expression and hold promise for therapeutic use where persistent transgene expression is required. To characterize the widely-observed silencing process affecting expression of foreign DNA in mammals, we used a system in which mouse liver presented with either plasmid or Minicircle consistently silences plasmid but not Minicircle expression. We found that preferential silencing of plasmid DNA occurs at a nuclear stage that precedes transport of mRNA to the cytoplasm, evident from a consistent >25-fold Minicircle/plasmid transcript difference observed in both nuclear and total RNA. Among possible mechanisms of nuclear silencing, our data favor chromatin-linked transcriptional blockage rather than targeted degradation, aberrant processing, or compromised mRNA transport. In particular, we observe dramatic enrichment of H3K27 trimethylation on plasmid sequences. Also, it appears that Pol II can engage the modified plasmid chromatin, potentially in a manner that is not productive in the synthesis of high levels of new transcript. We outline a scenario in which sustained differences at the chromatin level cooperate to determine the activity of foreign DNA.
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the extragenic spacer length between the 5 and 3 ends of the transgene expression cassette affects transgene silencing from plasmid based vectors
Molecular Therapy, 2012Co-Authors: Feijie Zhang, Andrew Fire, Mark A. KayAbstract:In quiescent tissues, Minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5′ and 3′ ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ≥1 kb in length resulted in transgene silencing while shorter spacers, ≤500 bp exhibited similar transgene expression patterns to conventional Minicircle DNA vectors. In contrast, when the ≥1 kb random DNA (RD) sequences were expressed as part of the 3′-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated.
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Generation of adult human induced pluripotent stem cells using nonviral Minicircle DNA vectors
Nature Protocols, 2011Co-Authors: Kazim H Narsinh, Mark A. Kay, Fangjun Jia, Robert C Robbins, Michael T LongakerAbstract:Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral Minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.
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A robust system for production of Minicircle DNA vectors
Nature biotechnology, 2010Co-Authors: Mark A. Kay, Zhi-ying ChenAbstract:Minicircle DNA vectors allow sustained transgene expression in quiescent cells and tissues. To improve Minicircle production, we genetically modified Escherichia coli to construct a producer strain that stably expresses a set of inducible Minicircle-assembly enzymes, ΦC31 integrase and I-SceI homing endonuclease. This bacterial strain produces purified Minicircles in a time frame and quantity similar to those of routine plasmid DNA preparation, making it feasible to use Minicircles in place of plasmids in mammalian transgene expression studies.
Fani Sousa - One of the best experts on this subject based on the ideXlab platform.
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the performance of Minicircle DNA versus parental plasmid in p53 gene delivery into hpv 18 infected cervical cancer cells
Nucleic Acid Therapeutics, 2021Co-Authors: Dalinda Eusebio, Joao A Queiroz, Fani Sousa, Ana Margarida Almeida, Joel Marques Alves, Claudio J Maia, ângela SousaAbstract:Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), thi...
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Minicircle DNA vaccine purification and e7 antigen expression assessment
Methods of Molecular Biology, 2021Co-Authors: Ana M Almeida, Dalinda Eusebio, Joao A Queiroz, Fani Sousa, ângela SousaAbstract:Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, Minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.
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Minicircle DNA the future for DNA based vectors
Trends in Biotechnology, 2020Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, ângela SousaAbstract:Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing.
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quality assessment of supercoiled Minicircle DNA by cadaverine modified analytical chromatographic monolith
Journal of Pharmaceutical and Biomedical Analysis, 2020Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, Urh Cernigoj, Ana C A SousaAbstract:Abstract Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1−25 μg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 μg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.
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the use of size exclusion chromatography in the isolation of supercoiled Minicircle DNA from escherichia coli lysate
Journal of Chromatography A, 2020Co-Authors: Ana Margarida Almeida, Dalinda Eusebio, Joao A Queiroz, Fani Sousa, Ana C A SousaAbstract:Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.
Joao A Queiroz - One of the best experts on this subject based on the ideXlab platform.
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the performance of Minicircle DNA versus parental plasmid in p53 gene delivery into hpv 18 infected cervical cancer cells
Nucleic Acid Therapeutics, 2021Co-Authors: Dalinda Eusebio, Joao A Queiroz, Fani Sousa, Ana Margarida Almeida, Joel Marques Alves, Claudio J Maia, ângela SousaAbstract:Minicircle DNA (mcDNA) has been suggested as a vanguard technology for gene therapy, consisting of a nonviral DNA vector devoid of prokaryotic sequences. Unlike conventional plasmid DNA (pDNA), thi...
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Minicircle DNA vaccine purification and e7 antigen expression assessment
Methods of Molecular Biology, 2021Co-Authors: Ana M Almeida, Dalinda Eusebio, Joao A Queiroz, Fani Sousa, ângela SousaAbstract:Human papillomavirus (HPV ) has been extensively associated with the development of cervical cancer due to the expression of oncoproteins like E7. This protein can interfere with pRB tumor suppressor activity, enabling the uncontrolled proliferation of abnormal cells. DNA vaccines are known as the third-generation vaccines, providing the ability of targeting viral infections such as HPV in a preventive and therapeutic way. Although current strategies make use of plasmid DNA (pDNA) as the vector of choice to be used as a DNA vaccine, Minicircle DNA (mcDNA) has been proving its added value as a non-viral DNA vector by demonstrating higher expression efficiency and increased biosafety than the pDNA. However, due to its innovative profile, few methodologies have been explored and implemented for the manufacture of this molecule. This chapter describes the detailed procedures for the production, extraction, and purification of supercoiled E7-mcDNA vaccine, by using size-exclusion chromatography to obtain mcDNA with a purity degree which meets the regulatory agency criteria. Then, the assessment of E7 antigen expression through immunocytochemistry is also described.
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Minicircle DNA the future for DNA based vectors
Trends in Biotechnology, 2020Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, ângela SousaAbstract:Minicircle DNA (mcDNA) is a smaller and safer version of non-viral DNA vectors that results from a cutting-edge in vivo recombination process to excise prokaryotic sequences from plasmid DNA (pDNA). Considering the molecule's potential and increasing interest as a non-viral DNA-based therapeutic, biomanufacturing methodologies need to be improved, especially in downstream processing.
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quality assessment of supercoiled Minicircle DNA by cadaverine modified analytical chromatographic monolith
Journal of Pharmaceutical and Biomedical Analysis, 2020Co-Authors: Ana Margarida Almeida, Joao A Queiroz, Fani Sousa, Urh Cernigoj, Ana C A SousaAbstract:Abstract Minicircle DNA (mcDNA) is the ultimate non-viral DNA vector, presenting higher biosafety and therapeutic effect than conventional plasmid DNA (pDNA). However, given the similarity between mcDNA and its precursor, the parental plasmid (PP), analytical methodologies established for pDNA are unable to distinguish mcDNA from PP. Thus, a new need emerged for the implementation of suitable, rapid and non-expensive analytical methodologies for the characterization of mcDNA samples. Recently, our research group was able to develop a purification strategy for the isolation of supercoiled (sc) mcDNA resorting to cadaverine-modified monolith. Considering the promising results obtained with this strategy, a cadaverine-modified analytical monolith was prepared and explored for mcDNA quantification. Thus, a strategy of three-step increasing NaCl gradient was considered to first elute RNA/protein content, then isolate sc mcDNA and finally eliminate PP and other impurities still bounded to the matrix. A calibration curve was constructed with different sc mcDNA standards within a range of 1−25 μg/mL. Linearity, accuracy, precision and selectivity of this method were validated according to the international guidelines and the limit of detection and the lower limit of quantification were determined as 1 μg/mL. For the first time, to the best of our knowledge, an analytical method for mcDNA quantification is described. Besides ensuring the safety of mcDNA application by assessing the product purity, such methodology can be used in the future to control industrial mcDNA production and purification, perhaps aiding in the establishment of optimized and less expensive biotechnological operations.
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the use of size exclusion chromatography in the isolation of supercoiled Minicircle DNA from escherichia coli lysate
Journal of Chromatography A, 2020Co-Authors: Ana Margarida Almeida, Dalinda Eusebio, Joao A Queiroz, Fani Sousa, Ana C A SousaAbstract:Minicircle DNA (mcDNA) is the new cutting-edge technology which researchers have been exploring for gene therapy and DNA vaccination. Although it presents enormous advantages in comparison to conventional plasmid DNA regarding bioactivity and safety, its challenging isolation from parental plasmid and miniplasmid has been setting back its launching in biomedical sciences. In this work, it is demonstrated the use of a simple size exclusion chromatographic method for the isolation of supercoiled mcDNA. Sephacryl S-1000 SF matrix was explored under different conditions (flow, peak fractionation volume and sample loading) to achieve the best performance and retrieve a mcDNA sample devoid of other bacterial contaminants or plasmid species resultant from the recombination process. This isolation methodology resulted in 66.7% of mcDNA recovery with 98.1% of purity. In addition, to show the robustness of the method, the potential of using this matrix for the isolation of a larger mcDNA was also evaluated. Upon adjusting the flow or the column volume, the larger mcDNA molecule was also successfully isolated. Overall, a simple and effective strategy has been established for the isolation of supercoiled mcDNA, underlining the potential of size exclusion chromatography in mcDNA separation.
Oleg Tolmachov - One of the best experts on this subject based on the ideXlab platform.
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tightly wound miniknot vectors for gene therapy a potential improvement over supercoiled Minicircle DNA
Medical Hypotheses, 2010Co-Authors: Oleg TolmachovAbstract:Minimized derivatives of bacterial plasmids with removed bacterial backbones are promising vectors for the efficient delivery and for the long-term expression of therapeutic genes. The absence of the bacterial plasmid backbone, a known inducer of innate immune response and a known silencer of transgene expression, provides a partial explanation for the high efficiency of gene transfer using minimized DNA vectors. Supercoiled Minicircle DNA is a type of minimized DNA vector obtained via intra-plasmid recombination in bacteria. Minicircle vectors seem to get an additional advantage from their physical compactness, which reduces DNA damage due to the mechanical stress during gene delivery. An independent topological means for DNA compression is knotting, with some knotted DNA isoforms offering superior compactness. I propose that, firstly, knotted DNA can be a suitable compact DNA form for the efficient transfection of a range of human cells with therapeutic genes, and, secondly, that knotted minimized DNA vectors without bacterial backbones ("miniknot" vectors) can surpass supercoiled Minicircle DNA vectors in the efficiency of therapeutic gene delivery. Crucially, while the introduction of a single nick to a supercoiled DNA molecule leads to the loss of the compact supercoiled status, the introduction of nicks to knotted DNA does not change knotting. Tight miniknot vectors can be readily produced by the direct action of highly concentrated type II DNA topoisomerase on Minicircle DNA or, alternatively, by annealing of the 19-base cohesive ends of the minimized vectors confined within the capsids of Escherichia coli bacteriophage P2 or its satellite bacteriophage P4. After reaching the nucleoplasm of the target cell, the knotted DNA is expected to be unknotted through type II topoisomerase activity and thus to become available for transcription, chromosomal integration or episomal maintenance. The hypothesis can be tested by comparing the gene transfer efficiency achieved with the proposed miniknot vectors, the Minicircle vectors described previously, knotted plasmid vectors and standard plasmid vectors. Tightly-wound miniknots can be particularly useful in the gene administration procedures involving considerable forces acting on vector DNA: aerosol inhalation, jet-injection, electroporation, particle bombardment and ultrasound DNA transfer.
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tightly wound miniknot vectors for gene therapy a potential improvement over supercoiled Minicircle DNA
Medical Hypotheses, 2010Co-Authors: Oleg TolmachovAbstract:Summary Minimized derivatives of bacterial plasmids with removed bacterial backbones are promising vectors for the efficient delivery and for the long-term expression of therapeutic genes. The absence of the bacterial plasmid backbone, a known inducer of innate immune response and a known silencer of transgene expression, provides a partial explanation for the high efficiency of gene transfer using minimized DNA vectors. Supercoiled Minicircle DNA is a type of minimized DNA vector obtained via intra-plasmid recombination in bacteria. Minicircle vectors seem to get an additional advantage from their physical compactness, which reduces DNA damage due to the mechanical stress during gene delivery. An independent topological means for DNA compression is knotting, with some knotted DNA isoforms offering superior compactness. I propose that, firstly, knotted DNA can be a suitable compact DNA form for the efficient transfection of a range of human cells with therapeutic genes, and, secondly, that knotted minimized DNA vectors without bacterial backbones ("miniknot" vectors) can surpass supercoiled Minicircle DNA vectors in the efficiency of therapeutic gene delivery. Crucially, while the introduction of a single nick to a supercoiled DNA molecule leads to the loss of the compact supercoiled status, the introduction of nicks to knotted DNA does not change knotting. Tight miniknot vectors can be readily produced by the direct action of highly concentrated type II DNA topoisomerase on Minicircle DNA or, alternatively, by annealing of the 19-base cohesive ends of the minimized vectors confined within the capsids of Escherichia coli bacteriophage P2 or its satellite bacteriophage P4. After reaching the nucleoplasm of the target cell, the knotted DNA is expected to be unknotted through type II topoisomerase activity and thus to become available for transcription, chromosomal integration or episomal maintenance. The hypothesis can be tested by comparing the gene transfer efficiency achieved with the proposed miniknot vectors, the Minicircle vectors described previously, knotted plasmid vectors and standard plasmid vectors. Tightly-wound miniknots can be particularly useful in the gene administration procedures involving considerable forces acting on vector DNA: aerosol inhalation, jet-injection, electroporation, particle bombardment and ultrasound DNA transfer.
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recet driven chromosomal gene targeting to generate a reca deficient escherichia coli strain for cre mediated production of Minicircle DNA
BMC Biotechnology, 2006Co-Authors: Oleg Tolmachov, Iwona Palaszewski, Brian W Bigger, Charles CoutelleAbstract: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. We describe here the construction of the RecA-deficient Minicircle DNA producer Escherichia coli HB101Cre with a chromosomally located Cre recombinase gene under the tight control of the araC regulon. The Cre gene expression cassette was inserted into the chromosomal lacZ gene by creating transient homologous recombination proficiency in the recA- strain HB101 using plasmid-born recET genes and homology-mediated chromosomal "pop-in, pop-out" of the plasmid pBAD75Cre containing the Cre gene and a temperature sensitive replication origin. Favourably for the Cre gene placement, at the "pop-out" step, the observed frequency of RecET-led recombination between the proximal regions of homology was 10 times higher than between the distal regions. Using the Minicircle producing plasmid pFIXluc containing mutant loxP66 and loxP71 sites, we isolated pure Minicircle DNA from the obtained recA- producer strain HB101Cre. The Minicircle DNA preparation consisted of monomeric and, unexpectedly, also multimeric Minicircle DNA forms, all containing the hybrid loxP66/71 site 5'-TACCGTTCGT ATAATGTATG CTATACGAAC GGTA-3', which was previously shown to be an inefficient partner in Cre-mediated recombination. Using transient RecET-driven recombination we inserted a single copy of the araC controlled Cre gene into the lacZ gene on the chromosome of E. coli recA- strain HB101. The resultant recA- Minicircle DNA producer strain HB101Cre was used to obtain pure Minicircle DNA, consisting of monomeric and multimeric Minicircle forms. The obtained recA- Minicircle DNA producer strain is expected to decrease the risk of undesired deletions within Minicircle producer plasmids and, therefore, to improve production of the therapeutic Minicircle vectors.