Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli.

Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.

Role of DNA topology in uptake of polyplex molecules by dendritic cells.

Dendritic cells (DCs) are an attractive target for DNA vaccines as they are potent antigen presenting cells. This study demonstrated how non-viral gene delivery to DCs involving complexes of poly-l-lysine (PLL) and plasmid DNA (pDNA) (polyplexes) showed dependence on DNA vector topology. DNA topology is of importance from both production and regulatory viewpoints. In our previous study with CHO cells we demonstrated that polyplex uptake was dependent on DNA topology whereby complexes containing supercoiled (SC) pDNA were smaller, more resistant to nucleases and more effectively condensed by PLL than open circular (OC) and linear-pDNA complexes. In this study polyplex uptake in DCs was measured qualitatively and quantitatively by confocal microscopy along with gene expression studies and measurement of DC phenotype. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Quantification studies revealed that by 1h following uptake 15% (±2.59% relative standard error [RSE]) of SC-pDNA polyplexes were identified to be associated (fluorescent co-localisation) with the nucleus, in comparison to no nuclear association identified for OC- and linear-pDNA complexes. By 48 h following uptake, 30% (±1.82% RSE) of SC-pDNA complexes associated with the nucleus in comparison to 16% (±4.40% RSE) and 12% (±6.97% RSE) of OC- and linear-pDNA polyplexes respectively. Confocal microscopy images showed how DNA and PLL remained associated following uptake by dual labelling. Polyplex (containing 20 µg pDNA) gene expression (plasmid encoded lacZ [ß-galactosidase] reporter gene) in DCs was greatest for SC-pDNA polyplexes at 14.12% unlike that of OC- (9.59%) and linear-pDNA (7.43%). DCs express cell surface markers which contribute towards antigen presentation. Polyplex gene expression did not alter DC phenotype through surface marker expression. This may be due to the pDNA dose employed (20µg) as other studies have used doses as high as 200 µg pDNA to induce DC phenotypic changes. Although no change in DC phenotype occurred, this could be advantageous in terms of biocompatibility. Collectively these results indicate that DNA topology is an important parameter for DC vector design, particularly pDNA in the SC conformation in regards to DNA vaccination studies.

The impact of DNA topology on polyplex uptake and transfection efficiency in mammalian cells.

The effect of DNA vector topology when complexed to poly-l-lysine (PLL) and its quantification in transfection efficiency has not been fully addressed even though it is thought to be of importance from both production and regulatory viewpoints. This study investigates and quantifies cell uptake followed by transfection efficiency of PLL:DNA complexes (polyplexes) in Chinese hamster ovary (CHO) cells and their dependence on DNA topology. PLL is known for its ability to condense DNA and serve as an effective gene delivery vehicle. Characterization of PLL conjugated to a 6.9kb plasmid was carried out. Dual labeling of both the plasmid DNA (pDNA) and PLL enabled quantitative tracking of the complexed as well as dissociated elements, within the cell, and their dependence on DNA topology. Polyplex uptake was quantified by confocal microscopy and image analysis. Supercoiled (SC) pDNA when complexed with PLL, forms a polyplex with a mean diameter of 139.06nm (±0.84% relative standard error [RSE]), whereas open circular (OC) and linear-pDNA counterparts displayed mean diameters of 305.54 (±3.2% RSE) and 841.5nm (±7.2% RSE) respectively. Complexes containing SC-pDNA were also more resistant to nuclease attack than its topological counterparts. Confocal microscope images reveal how the PLL and DNA remain bound post transfection. Quantification studies revealed that by 1h post transfection 61% of SC-pDNA polyplexes were identified to be associated with the nucleus, in comparison to OC- (24.3%) and linear-pDNA polyplexes (3.5%) respectively. SC-pDNA polyplexes displayed the greatest transfection efficiency of 41% which dwarfed that of linear-pDNA polyplexes of 18.6%. Collectively these findings emphasize the importance of pDNA topology when complexed with PLL for gene delivery with the SC-form being a key pre-requisite.