Transcript-activated collagen matrix as sustained mRNA delivery system for bone regeneration.

Transcript therapies using chemically modified messenger RNAs (cmRNAs) are emerging as safe and promising alternatives for gene and recombinant protein therapies. However, their applications have been limited due to transient translation and relatively low stability of cmRNAs compared to DNA. Here we show that vacuum-dried cmRNA-loaded collagen sponges, termed transcript activated matrices (TAMs), can serve as depots for sustained delivery of cmRNA. TAMs provide steady state protein production for up to six days, and substantial residual expression until 11days post transfection. Another advantage of this technology was nearly 100% transfection efficiency as well as low toxicity in vitro. TAMs were stable for at least 6months at room temperature. Human BMP-2-encoding TAMs induced osteogenic differentiation of MC3T3-E1 cells in vitro and bone regeneration in a non-critical rat femoral bone defect model in vivo. In summary, TAMs are a promising tool for bone regeneration and potentially also for other applications in regenerative medicine and tissue engineering.

Aerosolized BC-819 inhibits primary but not secondary lung cancer growth.

Despite numerous efforts, drug based treatments for patients suffering from lung cancer remains poor. As a promising alternative, we investigated the therapeutic potential of BC-819 for the treatment of lung cancer in mouse tumor models. BC-819 is a novel plasmid DNA which encodes for the A-fragment of Diphtheria toxin and has previously been shown to successfully inhibit tumor growth in human clinical study of bladder carcinoma. In a first set of experiments, we examined in vitro efficacy of BC-819 in human lung cancer cell-lines NCI-H460, NCI-H358 and A549, which revealed >90% reduction of cell growth. In vivo efficacy was examined in an orthotopic mouse xenograft lung cancer model and in a lung metastasis model using luminescent A549-C8-luc adenocarcinoma cells. These cells resulted in peri- and intra-bronchiolar tumors upon intrabronchial application and parenchymal tumors upon intravenous injection, respectively. Mice suffering from these lung tumors were treated with BC-819, complexed to branched polyethylenimine (PEI) and aerosolized to the mice once per week for a period of 10 weeks. Using this regimen, growth of intrabronchially induced lung tumors was significantly inhibited (p = 0.01), whereas no effect could be observed in mice suffering from lung metastasis. In summary, we suggest that aerosolized PEI/BC-819 is capable of reducing growth only in tumors arising from the luminal part of the airways and are therefore directly accessible for inhaled BC-819.

Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.

Aerosol gene delivery holds great therapeutical potential for many inherited and acquired pulmonary diseases. The physical instability of aqueous suspensions of non-viral vector complexes is a major limitation for their successful application. In this study, we investigated dry powder aerosols as novel gene vector formulations for gene transfer in vitro and murine lungs in vivo. Lyophilization was used to produce dry powder cakes followed by powderization to produce dry powder aerosols. Different sugars, namely lactose, sucrose and trehalose, were tested as lyoprotectants for gene delivery complexes consisting of branched polyethylenimine 25 kDa and plasmid DNA. Biophysical particle characterization demonstrated that lyophilization and powderization in the presence of lyoprotectants were well tolerated. In vitro transfection efficiency remained unaffected by the choice of lyoprotectant and subsequent lyophilization and/or powderization. In vivo screening of powderized samples, by applying the powder with an insufflator, resulted in highest gene expression with lactose as lyoprotectant. Delivering a plasmid coding for murine erythropoietin together with lactose as lyoprotectant resulted in increased blood hematocrit values post application thereby demonstrating the potential of dry powder aerosol as a promising method for pulmonary gene delivery.

Optimization of Streptomyces bacteriophage phi C31 integrase system to prevent post integrative gene silencing in pulmonary type II cells.

phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.

Transgene expression of transfected supercoiled plasmid DNA concatemers in mammalian cells.

BACKGROUND: Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers. METHODS: Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry. RESULTS: For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (E(gene)) and plasmid expression efficiency (E(plasmid)) by determining the ratio of the EGFP MFI of the transfected cells to TOTO-1 MFI per nucleus on the single cell level. E(gene) and E(plasmid) were significantly 1.6-and 3.5-fold higher for EGFP-dimer than for EGFP-monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFP-monomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation. CONCLUSIONS: We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells.

Cell type differences in activity of the Streptomyces bacteriophage phiC31 integrase.

Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery.

BACKGROUND: Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 integrase in murine lungs. METHODS: Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR). RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver. CONCLUSIONS: These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.

Targeting of the glucocorticoid hormone receptor with plasmid DNA comprising glucocorticoid response elements improves nonviral gene transfer efficiency in the lungs of mice.

BACKGROUND: It has been previously demonstrated that plasmid DNA transport into the nucleus could be increased by transcription factor binding. We chose the glucocorticoid responsive element (GRE) which binds to the glucocorticoid receptor (GR), a transcription factor which is shuttled into the nucleus upon ligand binding such as dexamethasone. METHODS: We cloned two, four, and eight repetitive sequences of the GRE into the reporter plasmid pEGFPLuc. Binding of the pEGFPLuc-GRE to the GR was examined by electrophoretic mobility shift assay (EMSA) experiments. GR expression in bronchiolar and alveolar epithelial cells was confirmed by Western blotting. Intracellular trafficking of GR was examined using a fusion protein of cyano-fluorescent protein (CFP) and GR. Transfection efficiencies of pEGFPLuc compared to pEGFPLucGRE(2-8) were examined in vitro and in vivo upon tail vein injection of cationic liposome gene vectors containing dexamethasone (safeplexes) and aerosol application of polyethylenimine (PEI)-pDNA particles. RESULTS: Binding of GRE containing plasmids to the GR was shown in EMSA experiments and intranuclear shuttling of CFP-GR after ligand stimulation was confirmed. Enhanced gene transfer efficiency of pEGFPLucGRE(2) in vitro was only observed on confluent cells. A 2.5-fold increase in gene expression in the lungs of mice after tail vein injection of pEGFPLucGRE(2) complexed with safeplexes compared with pEGFPLuc was observed. PEI-mediated aerosol gene delivery of pEGFPLucGRE(2) was 4.7-fold higher than pEGFPLuc only after intraperitoneal dexamethasone. CONCLUSION: The results suggest that inclusion of GRE sequences into plasmid DNA vectors combined with dexamethasone application could improve transgene expression in the lungs in vivo.

Influence of ozone on litter quality and its subsequent effects on the initial structure of colonizing microbial communities.

Ozone is considered as the main factor in air pollution related to a decline of forest in North America and Europe. In the present study, the effect of changed litter quality, due to ozone stress to trees, on the microbial communities colonizing the subsequent litter was investigated. Litter bag technique using beech and spruce litter from ozone-stressed and control trees, was combined with 16S and 18S rRNA-based fingerprinting methods and cloning to characterize phylogenetic diversity. Litter bags were incubated for 2 and 8 weeks in a beech-spruce mixed forest. Differences between the structure of microbial communities colonizing control and ozone-exposed litter were evident by fingerprints of 16S and 18S rRNA RT-PCR products. RT-PCR products, from litter degraded for 8 weeks, were cloned to identify the bacterial and fungal groups. Clones similar to members of Actinobacteria dominated the bacterial libraries, whereas effects of changed litter quality were mainly observed for the Proteobacteria. Fungal libraries were dominated by clones similar to Ascomycota members. Reduced proportion of clones similar to Basidiomycota and Zygomycota in library from ozone-stressed spruce trees and Chytridiomycota from ozone-stressed beech trees was observed when compared to their control counterparts. As hypothesized, changed litter quality due to elevated O3 did influence the structure of litter-colonizing microbial communities. However, these differences were not as pronounced as those between the two plant species.

Gene therapy of surfactant protein B deficiency.

Surfactant protein B (SP-B) is encoded by a single gene, and the mature protein is expressed by alveolar type II epithelial (ATII) cells of the lungs. Studies in transgenic mice and its hereditary deficiency in humans have established its indispensable role in postnatal survival. An established gene therapy regime for treating SP-B deficiency could overcome the limitations of surfactant replacement therapies or lung transplantation. Among the various viral and non-viral gene delivery tools available, only adenoviral vectors have been tested for delivering SP-B cDNA to the lungs of animal models. This review discusses the various vectors that are available for delivering therapeutic genes into ATII cells.

Microbial colonization of beech and spruce litter--influence of decomposition site and plant litter species on the diversity of microbial community.

The present study was conducted to investigate the effect of decomposition site and plant litter species on the colonizing microbial communities. For this, litter bag technique using beech and spruce litter was combined with RNA-based fingerprinting and cloning. Litter bags were incubated for 2 and 8 weeks in the Ah horizon of beech and beech-spruce mixed forest sites. Although sugars and starch were rapidly lost, lignin content increased by more than 40% for beech and more than doubled for spruce litter at both soil sites at the end of the experiment. Denaturing gradient gel electrophoresis analysis of 16S and 18S rRNA RT-PCR products was used for screening of differences between bacterial and fungal communities colonizing the two litter types. Development of the microbial community over time was observed to be specific for each litter type and decomposition site. RT-PCR products from both litter types incubated in beech-spruce mixed forest site were also cloned to identify the bacterial and fungal colonizers. The 16S rRNA clone libraries of beech litter were dominated by gamma-proteobacterial members, whereas spruce libraries were mainly composed of alpha-, beta-, and gamma-proteobacterial members. Ascomycota members dominated the 18S rRNA clone libraries. Clones similar to Zygomycota were absent from spruce, whereas those similar to Basidiomycota and Glomeromycota were absent from beech libraries. Selective effects of litter quality were observed after 8 weeks. The study provides an insight into the bacterial and fungal communities colonizing beech and spruce litter, and the importance of litter quality and decomposition site as key factors in their development and succession.

Diversity of transcripts of nitrite reductase genes (nirK and nirS) in rhizospheres of grain legumes.

Transcription of the nirK and nirS genes coding for dissimilatory bacterial nitrite reductases was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from rhizosphere samples of three economically important grain legumes at maturity: Vicia faba, Lupinus albus, and Pisum sativum. The nirK gene and transcripts could be detected in all the rhizosphere samples. In contrast, nirS could not be detected. Sampling variations were analyzed by comparing denaturing gradient gel electrophoresis profiles derived from nirK RT-PCR products. High similarity was observed between the replicates, and so one representative product per legume was cloned. Clones with the correct insert size were screened by restriction fragment length polymorphism by using the restriction enzyme MspI. The clones could be distributed into 12 different patterns. Patterns 1, 3, 4, 5, and 7 were common in clone libraries of the three rhizosphere types under study. Patterns 2, 9, 10, and 11 were absent from Pisum rhizospheres, while patterns 6, 8, and 12 were absent from the Vicia library. Pattern 1, which was the most dominant in the Vicia and Lupinus libraries, constituted about 25% of all clones. The Lupinus library had clones representing all 12 patterns, indicating it to be the most diverse among the three. Clones representative of each pattern were sequenced. All patterns grouped together forming a distinct cluster, which was divergent from previously described nirK sequences in the database. The study revealed a hitherto unknown diversity of denitrifiers in legume rhizospheres. A plant-dependent rhizosphere effect on the transcripts of a gene was evident.