Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.     

Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.     

Which mechanism for nuclear import of plasmid DNA complexed with polyethylenimine derivatives?

BACKGROUND: To investigate the nuclear import mechanism of plasmid/polyethylenimine (PEI) derivative complexes and the putative nuclear targeting of therapeutic genes by the use of oligosaccharides, we have studied the nuclear import of plasmid DNA complexed either with PEI or with lactosylated PEI (Lac-PEI) in cystic fibrosis human airway epithelial cells ( summation operatorCFTE29o- cells). METHODS AND RESULTS: Cells were synchronized by a double-thymidine block protocol and gene transfer efficiency was evaluated: Lac-PEI- and PEI-mediated gene transfer was greatly increased when cells have undergone mitosis during the course of transfection. However, both types of complexes were able to transfect some growth-arrested cells. When the nuclear import of plasmid/Lac-PEI or plasmid/unsubstituted PEI complexes was studied in digitonin-permeabilized cells, the nuclear uptake of both types of complexes did not follow the classic pathway of nuclear localization sequence (NLS)-containing proteins and lactose residues did not act as a nuclear localization signal. CONCLUSIONS: Our results show that for complexes made with PEI derivatives, the major route for plasmid DNA nuclear entry is a passive nuclear importation during mitosis when the nuclear membrane temporarily breaks down. However, albeit to a lesser extent as that observed in dividing cells, a plasmid DNA importation also occurs in nondividing cells by a yet unknown mechanism.     

DNA nanoparticles: detection of long-term transgene activity in brain using bioluminescence imaging

In this study, we used bioluminescence imaging (BLI) to track long-term transgene activity following the transfection of brain cells using a nonviral gene therapy technique. Formulations of deoxyribonucleic acid (DNA) combined with 30-mer lysine polymers (substituted with 10 kDa polyethylene glycol) form nanoparticles that transfect brain cells in vivo and produce transgene activity. Here we show that a single intracerebral injection of these DNA nanoparticles (DNPs) into the rat cortex, striatum, or substantia nigra results in long-term and persistent luciferase transgene activity over an 8- to 11-week period as evaluated by in vivo BLI analysis, and single injections of DNPs into the mouse striatum showed stable luciferase transgene activity for 1 year. Compacted DNPs produced in vivo signals 7- to 34-fold higher than DNA alone. In contrast, ex vivo BLI analysis, which is subject to less signal quenching from surrounding tissues, demonstrated a DNP to DNA alone ratio of 76- to 280-fold. Moreover, the ex vivo BLI analysis confirmed that signals originated from the targeted brain structures. In summary, BLI permits serial analysis of luciferase transgene activity at multiple brain locations following gene transfer with DNPs. Ex vivo analysis may permit more accurate determination of relative activities of gene transfer vectors.     

Cloning and Functional Expression of a Novel Human Connexin-25 Gene

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Transport of polymeric nanoparticle gene carriers in gastric mucus

Nanoparticle transport through mucosal barriers is often restricted owing to mucoadhesion and the highly viscoelastic nature of mucus gels, which may limit efficient drug and gene delivery. We formulated sub-200 nm particulates from poly(d,l-lactic-co-glycolic) acid (PLGA) and the cationic surfactant dimethyldioctadecylammonium bromide (DDAB). Subsequently, anionic DNA was condensed to the surface to obtain gene carriers with transfection rates 50-fold higher than those of naked DNA in vitro. Using the method of multiple particle tracking (MPT), we measured the transport rates of dozens of individual PLGA-DDAB/DNA nanoparticles in real time in reconstituted pig gastric mucus (PGM) that possessed physiologically relevant rheological properties. The average transport rate of PLGA-DDAB/DNA nanoparticles was 10-fold higher than those of similar size polystyrene nanoparticles. Improved transport rates, stability in mucus, and ability to transfect cells make PLGA-DDAB/DNA nanoparticles candidates for mucosal DNA vaccines and gene therapy.     

Tracking the pathway of calcium phosphate/DNA nanoparticles during cell transfection by incorporation of red-fluorescing tetramethylrhodamine isothiocyanate–bovine serum albumin into these nanoparticles

Calcium phosphate nanoparticles were prepared by precipitation from water and were then functionalized by DNA. These particles are taken up by living cells and function as gene transfer agents, i.e., the DNA is brought into a cell’s nucleus and is incorporated there into the cell’s genome (transfection). DNA which encodes for enhanced green fluorescent protein leads to green fluorescence of successfully transfected cells. By adding the red-fluorescing marker tetramethylrhodamine isothiocyanate–bovine serum albumin (TRITC-BSA) to the nanoparticles, their pathway into the cell and within the cell could be followed by fluorescence microscopy. A clear correlation between the uptake of nanoparticles and the efficiency of transfection was found. Aggregates of DNA/TRITC-BSA alone were not able to enter the cells, i.e., the inorganic nanoparticles are necessary as a carrier through the cell membrane.     

Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Efficient cold transfection of pea ferredoxin-NADP(H) oxidoreductase into rat hepatocytes

We describe the use of a non-viral, polyethylenimine-based vector to transfect rat hepatocytes preserved under hypothermic storage. DNA sequences encoding Escherichia coli beta-galactosidase and pea ferredoxin-NADP(H) oxidoreductase (FNR), cloned into plasmids pCH110 and pKM4 respectively, were used. FNR was detected in the liver of animals transplanted with transfected cells; no reactivity was observed in endogenous parenchyma. The expression of the transgene was transient as it was detectable up to 96 h subsequently declining to undetectable levels. In contrast to non-transfected cells, the engraftment of FNR-positive cells was not associated with inflammatory reaction. The percentage of FNR-positive implanted hepatocytes was at least five times higher than the original transfection efficiency measured in vitro, while the percentage of beta-galactosidase-positive cells was similar for both methods. These data indicate that the transfection system is effective in the transfer of plasmid DNA into hepatocytes under cold preservation and suggest the advantage of pKM4-transfected hepatocytes on engraftment in the recipient parenchyma.     

Study on the Multidrug Resistance 1 Gene Transfection Efficiency Using Adenovirus Vector Enhanced by Ultrasonic Microbubbles In Vitro

As gene delivery reagents, microbubbles have been successfully used in combination with ultrasound. Shock wave exposure has been shown to transfect cells with naked DNA in vitro, but it has not been tested whether the addition of microbubbles would enhance DNA uptake with adenovirus vector. Therefore, the aim of this study was to study the efficacy and safety of multidrug resistance 1 (MDR1) gene transfer into the bone marrow mononuclear cells of rabbits using adenovirus vector enhanced by ultrasound with microbubbles in vitro. The transfection rate of the MDR1 gene was significantly increased by ultrasound microbubbles with adenovirus. After ultrasonic irradiation, there were transient holes in the cell membrane, which disappeared after irradiation by ultrasound for 24 h. The temporary swelling of the organelles was reversible. Our in vitro findings conclusively demonstrate that the exogenous MDR1 gene transfer into the mononuclear cells of rabbits with adenovirus vector was enhanced by the ultrasonic microbubbles and this transfection technique is safe.     

Caecum: A Potential Site for Studying Gene Transfer in vivo

Abstract

Over the past years we have designed and synthesized a new class of synthetic vectors called phosphonolipids and then shown their ability to transfect lungs cells of mice with efficiency. One of them, GLB73, gave high levels of transfection. In the study reported here, we explored the potential of caecum as an alternative site for studying the feasibility of gene transfer in this site. Transfections were performed by using two reporter genes encoding for (igalactosidase and luciferase; transfection activity was assessed using two tests: chemiluminescent and cytofiuorimetric assays. The results obtained showed successful gene transfer into the caecum: up to 27% cells were LacZ+ with a mean of 11%; the maximum of efficiency was also observed 3 days after transfection which then decreased until day 7. Our lipoplex was 8-fold more efficient than the naked DNA (Mann Whitney test; p = 0.03). Moreover, we were able to visually follow the uptake of lipoplexes by enterocytes from 30 mn to 3 days post transfection.

So, this study constituted an encouraging first step in the assessment of the caecum as a potential model for gene transfer. In the near future, further electrophysiological studies using the gene of interest as CFTR gene should be performed in the caecum.     

Editorial,Introduction and Acknowledgements

Gap junctions arc intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Cloning and functional expression of a novel human connexin-25 gene

Gap junctions are intercellular, water-filled channels composed of transmembrane proteins called connexins, six of which are arranged radially and dock with six homologous proteins in an adjacent cell to form an approximate 16 A pore. Through this pore cell-to-cell transfer of small water-soluble molecules up to about 1000 daltons occurs along concentration gradients. Connexins comprise a multigene family that share consensus sequences in the trans-membrane domains and the first and second extracellular loops. Comparison of the protein sequences of known human connexins with the draft nucleotide sequence of the human genome revealed two clones from chromosome 6 which showed strong similarity to highly conserved connexin sequences. Detailed analysis revealed the presence of a 672 nt open reading frame in these clones, encoding a 223 amino acid polypeptide with a predicted molecular weight of about 25 kD. This is smaller than other known human connexins. The ORF of the potential connexin25 was amplified by semi-nested PCR using human genomic DNA as a template. To confirm that this new gene encodes a connexin, Cx25 was transfected into a gap junction deficient subclone of the human HeLa cell line. After selection of transformants, cells were microinjected with the fluorescent dye Lucifer yellow. Transfectants but not controls successfully transferred dye, demonstrating that this new gene encodes a functional connexin.     

Avian retrovirus integrase-enhanced transgene integration into mammalian cell DNA in vivo

Systems for introducing DNA genes-of-interest into mammalian cellular genomes have ranged from the use of different physical techniques to viruses including retroviruses. We have developed a microinjection method for an efficient and permanent integration of a DNA transgene into the cell genome by use of the retrovirus integrase. A 3.0-kb linear DNA fragment containing an internal herpes simplex virus thymidine kinase gene (tk) with flanking avian retrovirus U5 and U3 terminal attachment sites (U5-pgk/tk-U3) recognized by the integrase was constructed. The other donor, a 3.3-kb linear DNA fragment containing the same gene (pgk/tk) flanked by ApaL1 restriction sites not recognized by integrase, was also produced. After assembly of integrase-transgene complexes on ice, the complexes were microinjected into the nucleus of human fibroblast cells (143Btk) containing a defective thymidine kinase. The number of hypoxanthine/aminopterin/thymidine (HAT)-resistant colonies produced upon microinjection of either naked DNA or the independently assembled integrase-transgene complexes were determined. Our data suggests that enhanced integration of U5-pgk/tk-U3 required the DNA attachment sites and co-delivery of integrase. The data was consistent with a direct role for both of these elements in producing an approximate 4-fold increase in the number of HAT-resistant colonies observed over microinjection of just naked U5-pgk/tk-U3 (P < 0.0001).     

E1A specifically enhances sensitivity to topoisomerase IIalpha targeting anticancer drug by up-regulating the promoter activity

DNA topoisomerases I and II (topo I and II) are nuclear enzymes involved in cellular replication and are targets for several anticancer drugs. We showed previously that E1A gene transfer enhanced the sensitivity of Ewing's sarcoma cells to the topo IIalpha targeting agents etoposide and Adriamycin in vitro and in vivo. To determine whether this effect was specific for topo IIalpha, we investigated the effect of E1A gene transfer on cell sensitivity to agents that target topo I and IIbeta. Transfecting TC71 human Ewing's sarcoma cells with an adenoviral vector containing the E1A gene enhanced their sensitivity to the topo IIalpha targeting agents etoposide (16-fold) and Adriamycin (8-fold). By contrast, E1A gene transfer did not affect cellular sensitivity to either amsacrine or camptothecin. Western blot analysis indicated that topo IIalpha protein levels increased 3.1-fold after E1A gene transfer, but topo I and IIbeta protein levels did not change. A plasmid containing topo IIalpha gene promoter with luciferase reporter gene was constructed to determine the effects of E1A gene transfer on the activity of the topo IIalpha promoter. E1A increased the activity of the topo IIalpha gene promoter by 3.5-fold relative to that of cells transfected with Ad-beta-gal. These results suggest that elevated topo IIalpha protein levels and enhanced sensitivity to topo IIalpha targeting agents were secondary to a direct effect of E1A on the topo IIalpha promoter. Combining E1A gene therapy with topo IIalpha targeting anticancer drugs may therefore have therapeutic benefit by increasing tumor cell sensitivity.     

一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功     

Identification of transfected cell types following non-viral gene transfer to the murine lung

BACKGROUND: Identification of the cell types transfected following gene transfer is an important factor in the selection of appropriate gene transfer agents (GTAs). Due to the relatively low gene expression mediated by non-viral GTAs, current methodologies for the detection and identification of transfected cells in the lung have proven insensitive and unreliable. We have investigated the use of the green fluorescent protein (GFP) to identify transfected cells in a mouse lung model. METHODS: Direct visualisation of GFP fluorescence in frozen histological sections was used in conjunction with a panel of cell type specific antibodies to investigate the distribution and level of gene expression in mouse lungs following instillation of non-viral GTAs. RESULTS: Despite considerable tissue autofluorescence, dose-dependent expression of GFP was detected following instillation of as little as 25 microg naked plasmid DNA (pDNA). Naked pDNA and pDNA complexed with polyethylenimine appeared to transfect mainly ciliated cells and Clara cells of the conducting airway, whereas expression mediated by pDNA complexed with the cationic lipid GL67 was found predominantly in type I pneumocytes. CONCLUSIONS: Direct visualisation of GFP expression was used to detect transfected cell types in the mouse lung. In contrast with observations made using beta-galactosidase as a reporter, gene expression from several non-viral GTAs was readily demonstrated and no false GFP-positive cells were ever detected in untreated lung tissues. Lung delivery of different GTAs resulted in GFP expression in different cell types, confirming the importance of identification of transfected cells when screening and selecting GTAs for disease targets.     

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.     

Factors mediating lipofection potency of a series of cationic phosphonolipids in human cell lines

A series of cationic liposomes known as cationic phosphonolipids (CPs) were evaluated as vehicles for in vitro gene transfer in K562 erythroleukemia cells and 5637 epithelial carcinoma cells. For each CP and target cell type examined, detailed analyses were performed to determine optimal transfection conditions (lipid/ DNA (+/-) charge ratio, amount of complexed episomal DNA, liposomal and lipoplex size, complexation medium and duration of complex-cell exposure time). Lipofection conditions were determined to be both cell- and lipid-type specific. Complexation medium critically affected transfection competence. The initial size of the liposome was not always predictive of lipofection potency. The lipid chemical composition had a strong impact upon lipofection efficiency; DOPE inclusion in the liposome formulations was found to affect the levels of transgene expression in a cell-dependent way. Notably, effective transgene expression was characterized by prominent plasmid nuclear incorporation. Human A gamma- and epsilon-globin transgene nuclear incorporation and expression in 5637 cells post GLB.391-mediated lipofection lends credence to its use as a vehicle of therapeutic transgene delivery.     

Gene transfer into adult rat spinal cord using naked plasmid DNA and ultrasound microbubbles

BACKGROUND: Although gene therapy might become a promising approach to treat spinal cord injury, the safety issue is a serious consideration in human gene therapy. Plasmid DNA transfer is safer than viral vectors, but the transfection efficiency is quite low. To overcome the problem, we applied the ultrasound microbubbles-mediated transfection method to the spinal cord in adult rats, since ultrasound microbubbles have been reported to be efficient to increase transfection efficiency in various tissues. METHODS: After exposing T9-10 spinal cord with a laminectomy, we injected a mixture of naked plasmid DNA and microbubbles into cerebrospinal fluid by lumbar puncture. Then, the T9-10 spinal cord was exposed to ultrasound. CONCLUSIONS: An ultrasound intensity of 0.4-0.5 W/cm2 significantly increased luciferase expression up to approximately 15-60-fold at the insonated level as compared to naked plasmid DNA alone. Luciferase activity could be detected at least up to 7 days after transfection, while the expression level was almost returned to undetectable level at 14 days after transfection. The transfected cells were mainly meningeal cells in the surface of insonated spinal cord. There was no obvious evidence of worsening of neurological deficits as compared to rats transfected with naked plasmid DNA alone or untransfected rats. Similarly, successful gene transfer was also achieved in the insonated T9-10 spinal cord after spinal cord injury. Overall, the present study demonstrated the feasibility of ultrasound microbubbles-mediated plasmid DNA transfer into the target level of the spinal cord.