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Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

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Transfection efficiency of lipoplexes in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
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Figure 6: Transfection efficiency of lipoplexes in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.

Mentions: The endosomal escape mechanism of DOTAP:DOPE-based lipoplexes is based on a different mechanism and results in the release of uncomplexed pDNA in the cytosol (47). This implies that, when the free pDNA reaches the nuclear membrane, TCHD should be able to induce its nuclear translocation, similar to the naked pDNA transfections. Therefore, we analyzed the effect of TCHD on the transfection efficiency of DOTAP:DOPE-based lipoplexes. The same incubation protocols and cells were used as in the experiments above. Incubating the cells with TCHD for 1 h immediately after incubation with the lipoplexes, indeed caused an increase in transfection efficiency in both Vero and A549 cells (Figure 6A and B). A maximal increase was observed when the Vero and A549 cells were incubated with 0.5 and 1.5% (w/v) TCHD, respectively. This increase is lower than observed with naked pDNA transfection, which can be attributed to cell division. Compared to naked pDNA transfection, transfection with lipoplexes introduces a higher amount of pDNA in the cytoplasm, which can translocate to the nucleus with higher probability either by entry through the pores or during cell division. Hence, the few extra copies that enter the nucleus after treating the cells with TCHD will not tremendously increase gene expression. In contrast, the amount of pDNA that reaches the cytoplasm and subsequently the nucleus is extremely low in case of naked pDNA transfection. Therefore, if TCHD can cause nuclear translocation of only one pDNA molecule, this effect is much more spectacular.Figure 6.


Nuclear accumulation of plasmid DNA can be enhanced by non-selective gating of the nuclear pore.

Vandenbroucke RE, Lucas B, Demeester J, De Smedt SC, Sanders NN - Nucleic Acids Res. (2007)

Transfection efficiency of lipoplexes in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919477&req=5

Figure 6: Transfection efficiency of lipoplexes in Vero (A) and A549 (B) cells post-incubated for 1 h with different TCHD concentrations. The asterisk (*) represents data that significantly differ (P < 0.05) from the data point 0% (w/v) TCHD.
Mentions: The endosomal escape mechanism of DOTAP:DOPE-based lipoplexes is based on a different mechanism and results in the release of uncomplexed pDNA in the cytosol (47). This implies that, when the free pDNA reaches the nuclear membrane, TCHD should be able to induce its nuclear translocation, similar to the naked pDNA transfections. Therefore, we analyzed the effect of TCHD on the transfection efficiency of DOTAP:DOPE-based lipoplexes. The same incubation protocols and cells were used as in the experiments above. Incubating the cells with TCHD for 1 h immediately after incubation with the lipoplexes, indeed caused an increase in transfection efficiency in both Vero and A549 cells (Figure 6A and B). A maximal increase was observed when the Vero and A549 cells were incubated with 0.5 and 1.5% (w/v) TCHD, respectively. This increase is lower than observed with naked pDNA transfection, which can be attributed to cell division. Compared to naked pDNA transfection, transfection with lipoplexes introduces a higher amount of pDNA in the cytoplasm, which can translocate to the nucleus with higher probability either by entry through the pores or during cell division. Hence, the few extra copies that enter the nucleus after treating the cells with TCHD will not tremendously increase gene expression. In contrast, the amount of pDNA that reaches the cytoplasm and subsequently the nucleus is extremely low in case of naked pDNA transfection. Therefore, if TCHD can cause nuclear translocation of only one pDNA molecule, this effect is much more spectacular.Figure 6.

Bottom Line: Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success.Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes.In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium.

ABSTRACT
One of the major obstacles in non-viral gene transfer is the nuclear membrane. Attempts to improve the transport of DNA to the nucleus through the use of nuclear localization signals or importin-beta have achieved limited success. It has been proposed that the nuclear pore complexes (NPCs) through which nucleocytoplasmic transport occurs are filled with a hydrophobic phase through which hydrophobic importins can dissolve. Therefore, considering the hydrophobic nature of the NPC channel, we evaluated whether a non-selective gating of nuclear pores by trans-cyclohexane-1,2-diol (TCHD), an amphipathic alcohol that reversibly collapses the permeability barrier of the NPCs, could be obtained and used as an alternative method to facilitate nuclear entry of plasmid DNA. Our data demonstrate for the first time that TCHD makes the nucleus permeable for both high molecular weight dextrans and plasmid DNA (pDNA) at non-toxic concentrations. Furthermore, in line with these observations, TCHD enhanced the transfection efficacy of both naked DNA and lipoplexes. In conclusion, based on the proposed structure of NPCs we succeeded to temporarily open the NPCs for macromolecules as large as pDNAs and demonstrated that this can significantly enhance non-viral gene delivery.

Show MeSH
Related in: MedlinePlus