The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases

BACKGROUND: Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease-rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease-resistant a priori. METHODS AND RESULTS: The hypothesis that a significant part of nuclease attack is directed towards certain labile sequences in a pDNA model (pVAX1/lacZ) was first tested. Homopurine-rich tracts in the bovine growth hormone polyadenylation signal (BGH poly A) were identified as labile sequences using S1 nuclease as a probe. Two pDNA variants were then created by replacing the BGH poly A region with the SV40 or a synthetic poly A signal. A study of plasmid degradation in eukaryotic cell lysates and mice plasma showed that the half-life of the supercoiled isoforms of the new vectors was always higher when compared with the control plasmid. An in vitro assay of the reporter beta-galactosidase in transfected CHO cells further showed that gene expression with the new pDNA variants was not affected negatively by the plasmid modifications. CONCLUSIONS: The replacement of labile sequences in plasmid DNA vectors improves resistance towards nuclease attack as shown by the increased half-lives of supercoiled plasmid isoforms incubated with endo/lysosomal, cytoplasmatic and blood plasma enzymes.     

Purification of plasmid DNA from Escherichia coli ferments using anion-exchange membrane and hydrophobic chromatography

A novel downstream bioprocess was developed to obtain purified plasmid DNA (pDNA) from Escherichia coli ferments. The intermediate recovery and purification of the pDNA in cell lysate was conducted using hollow-fiber tangential filtration and frontal anion-exchange membrane and elution hydrophobic chromatographies. The purity of the solutions of pDNA obtained during each process stage was investigated. The results show that the pDNA solution purity increased 30-fold and more than 99% of RNA in the lysate was removed during the process operations. The combination of membrane operations and hydrophobic interaction chromatography resulted in an efficient way to recover pDNA from cell lysates. A better understanding of membrane-based technology for the purification of pDNA from clarified E. coli lysate was developed in this research.     

Nondisruptive, sequence-specific coupling of fluorochromes to plasmid DNA

A method to attach a fluorochrome sequence-specifically to supercoiled plasmid DNA (pDNA) without perturbing transgene expression would provide an invaluable aid in a variety of applications requiring probes for the intracellular tracking of transfected pDNA. Here we report a method to couple commercially available fluorochromes covalently and sequence-specifically to pDNA using a peptide nucleic acid (PNA) as a linker molecule. The terminal cysteine thiol group on the PNA peptide backbone is reacted with a maleimide moiety on the fluorochrome to produce a fluorescent conjugate which is in turn hybridized to a plasmid expression vector containing an 11-bp target sequence. Spectroscopic evaluation and an electrophoretic mobility shift assay showed that the pDNA hybridized to one PNA-fluorochrome conjugate molecule. The fluorescence signal comigrated with pDNA on acrylamide gels, confirming the stable attachment of the fluorescent conjugate to the pDNA. The utility of one of the conjugates, PNA-Oregon green 488/pCMVbeta-DTS, to probe pDNA transport across the nuclear envelope, a significant barrier to gene transfer, was undertaken using a digitonin-permeabilized HeLa cell assay. The PNA-Oregon green 488/pCMVbeta-DTS conjugate is able to efficiently traverse the nuclear membrane of the permeabilized cells, accumulating in the nuclei within 30 min and reaching maximal levels by 1h. When transfected into HeLa cells, the PNA-Oregon green 488/pCMVbeta-DTS conjugate retained 55% of the native plasmid's biological activity, as determined by a beta-galactosidase assay. Thus, this method allows for the sequence-specific coupling of commercially available fluorochromes to DNA expression vectors while retaining biological function.     

Fluorometric determination of DNA and RNA in Chlamydomonas using ethidium bromide

By using the fluorescence enhancement of ethidium bromide bound to nuclei acid, a very rapid, simple and sensitive assay of DNA in the green alga Chlamydomonas has been devised. Total fluorescence (DNA + RNA) was determined by complex formation with ethidium bromide in a cell lysate made by mixing cell samples with lauroyl sarcosinate, EDTA and NaOH and incubating the mixture for 5 min at room temperature followed by neutralization. For determination of DNA the RNA was digested by incubating the cell sample in te alkaline lysis solution for 45 min at 60 degrees C followed by neutralization, and complex formation with ethidium bromide. Quenching of the fluorescence due to cellular pigments was corrected for using an internal DNA standard.     

Separation of supercoiled from open circular forms of plasmid DNA,and biological activity detection

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.     

Plasmid DNA purification

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA. The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i). different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii). the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii). the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins). We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy.     

一种体内标记转录RNA的新技术

目的:验证一种体内标记转录RNA的新技术,并用该技术观察一种长寿药物雷帕霉素对真核细胞HEK293中RNA合成的影响。方法:将尿嘧啶类似物5-乙炔尿苷(EU)和雷帕霉素加到HEK293细胞培养基中,共同孵育2h,然后在激光共聚焦显微镜下对EU标记的新合成RNA进行观察;用Image-pro plus软件对图像的荧光强度进行分析,获得反应荧光强度的平均光密度数值;用SPSS软件对数值进行统计分析。结果:激光共聚焦显微镜下,可见EU标记的新合成RNA主要分布在胞质和核仁中,以核仁中荧光最强;Image-pro plus软件和SPSS软件分析表明,加入雷帕霉素前后,细胞新合成的RNA平均光密度无明显差别。结论:EU是一种安全、简单、快速而灵敏的检测新合成RNA的新技术,用该技术检测表明长寿药物雷帕霉素不影响真核细胞HEK293中总RNA的合成。     

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

BACKGROUND: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. METHODS: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. RESULTS: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. CONCLUSIONS: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.     

SDF-1α基因与绿色荧光蛋白融合载体的构建及亚细胞定位

目的构建SDF-1α基因与绿色荧光蛋白的融合蛋白表达载体,进而观察SDF-1α基因编码蛋白在细胞内的定位情况。方法用EcoRI内切酶从pMD-T18一SDF-1α重组载体中酶切分离SDF-1α基因的完整ORF,构建pEGFP-C1-SDF-1α的融合表达载体,脂质体转染COS-7细胞,并在荧光显微镜下观察表达的融合蛋白。结果SDF-1α基因在COS-7细胞中高效表达,激光共聚焦的结果显示,SDF-1α基因定位在细胞质内。结论成功构建了pEGFP-C1-SDF-1α的融合表达载体,SDF-1α基因主要在细胞质中表达。     

Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei

This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.     

Changes in the nuclear polyamine content of chick erythrocytes during embryonic development.

The polyamine content of the circulating erythrocyte population in the embryonic chick was studied during its development. Total cellular polyamine content fell dramatically between 5 and 7 days of development, paralleling the decrease in metabolic activity exhibited by these cells. Nuclei were isolated from the erythrocytes by a non-aqueous technique, which not only eliminated the polyamine loss that occurred with aqueous isolation, but also prevented redistribution of the polyamines from the cytoplasm. Nuclear spermidine and spermine contents decreased markedly between 5 and 6 days of development from 31 to 10 pmol/microgram of DNA and from 33 to 18 pmol/microgram of DNA respectively. Thereafter the spermine content remained constant, but the spermidine content continued to decline. Good correlations between spermidine and RNA contents were observed in both cells and nuclei, and similarly between spermine and RNA contents in cells, but no such correlation was observed between spermine and RNA in nuclei.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter

BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. CONCLUSIONS: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.     

Inter-organ differences of the cytometric DNA content in mice: relation of the staining method

With one step DNA staining methods including cell membrane lysis and RNase treatment, we regularly observed a higher fluorescence emission in liver nuclei compared to bone marrow nuclei in C57BL/6 mice. Therefore this study was conducted in order to emphasize such a phenomenon in other organs and to assess if higher fluorescence emission was related to higher DNA content or staining procedure failure. Liver, bone marrow and testis were removed from Swiss, BDF and C57BL/6 mice. The following samples were prepared: 1) liver cells with TRBC (TRBC = Trout Red Blood Cells = internal standards), 2) bone marrow cells with TRBC, 3) testis cells with TRBC and 4) mixtures of liver, bone marrow and testis cells. The staining procedures were: A) one step pH 10 procedure described by Vindelov (Virchows Arch. B. Cell Path., 1977, 24, 227-242), B) same procedure with twice RNase concentration, C) first method with twice NP 40 concentration and D) three steps procedure including Trypsin and Spermine treatment (Vindelov et al., Cytometry, 1983, 3, 323-327). In protocols A, B and C, "Diploid cells/TRBC" ratio differed significantly between liver, bone marrow and testis nuclei. Moreover, 3 distinct populations of diploid cells were present in samples 4. In protocol D, "Diploid cells/TRBC" ratio were identical between liver, bone marrow and testis nuclei. In samples 4, only 1 population of diploid cells has been observed. This study shows that DNA stabilization by polyamine and protein degradation by protease could act on Propidium Iodide fixation and/or fluorescence emission, with significant differences according to the origin of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5alpha-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.     

Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposomes

BACKGROUND: Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection. METHODS: In order to combine the favorable features of both vectors, ternary complexes were prepared by adding cationic liposomes to plasmid DNA condensed by cationic detergents. The structure and physicochemical properties of these complexes were investigated by electron microscopy, quasi-elastic light scattering, gel electrophoresis and fluorescence techniques. These data were then correlated with the transfection efficiency and intracellular trafficking of the ternary complexes determined by luciferase gene expression and confocal microscopy, respectively. RESULTS: The ternary complexes were found to form small, homogeneous, globular, stable and positively charged particles with a highly dense and packed lamellar internal structure differing from the multilamellar structure (L(alpha)(C)) of the corresponding lipoplexes. In the presence of serum, the ternary complexes were more efficiently internalized into cells, less toxic and showed 20-fold higher transfection efficiency than lipoplexes. CONCLUSIONS: This study showed that small, monodisperse and highly stable complexes could be obtained by precompaction of DNA with cetyltrimethylammonium bromide, followed by addition of cationic lipids. The higher efficiency of the ternary complexes with respect to their corresponding lipoplexes was related to their internal structure which prevents their dissociation by serum proteins and allows efficient internalization in the target cells.     

Ribozyme-mediated RNA degradation in nuclei suspension.

Ribozymes containing 2'-fluoro- and 2'-amino-modified pyrimidine nucleosides in combination with terminal phosphorothioate linkages were targeted against HTLV-I tax RNA. In order to examine the activity of such chemically modified ribozymes in the nuclear environment, they were incubated with nuclei of a Tax-transformed mouse fibroblast cell line. Ribozyme cleavage of tax RNA was analyzed by the RNase protection assay. Comparison of the cleavage of tax RNA isolated nuclei with that of tax RNA present in nuclei suspension revealed a 30 times more efficient cleavage of the latter one. Pre-treatment with proteinase K and SDS abolished the enhancement of the ribozyme-mediated RNA cleavage. Catalytically inactive ribozymes did not yield any cleavage products. These results demonstrate an augmenting effect of nuclear proteins on the ribozyme-mediated RNA cleavage.     

Role of endonuclease G in exogenous DNA stability in HeLa cells

Endonuclease G (EndoG) is a well-conserved mitochondrial-nuclear nuclease with dual lethal and vital roles in the cell. The aim of our study was to examine whether EndoG exerts its nuclease activity on exogenous DNA substrates such as plasmid DNA (pDNA), considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four-day time course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus, targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances, it may nonspecifically cleave intracellular DNA regardless of its origin. These findings make it unlikely that targeting of EndoG is a viable strategy for improving the duration and level of transgene expression from nonviral DNA vectors in gene therapy efforts.