Limits...
Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery.

Matschke J, Bohla A, Maucksch C, Mittal R, Rudolph C, Rosenecker J - PLoS ONE (2012)

Bottom Line: We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method.The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo.This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany.

ABSTRACT
Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

Show MeSH
Analysing luciferase expression in lung homogenisates after nasal instillation of gene transfer agents.1, Ku702-NLS/PEI/DNA; 2, s1Ku702-NLS/PEI/DNA; 3, s2Ku702/PEI/DNA; 4, PEI/DNA. Gene vector complexes were formulated as follows: 30 µg DNA; NLS: ± ratio = 5; PEI: N/P ratio = 10. Luciferase activity was measured in lung homogenisates 24 h after application of gene vectors. Values between Ku702-NLS/PEI and PEI significantly different (p≤0.043; n = 4). Statistical analysis was performed using the Mann-Whithney-U test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3275586&req=5

pone-0024615-g004: Analysing luciferase expression in lung homogenisates after nasal instillation of gene transfer agents.1, Ku702-NLS/PEI/DNA; 2, s1Ku702-NLS/PEI/DNA; 3, s2Ku702/PEI/DNA; 4, PEI/DNA. Gene vector complexes were formulated as follows: 30 µg DNA; NLS: ± ratio = 5; PEI: N/P ratio = 10. Luciferase activity was measured in lung homogenisates 24 h after application of gene vectors. Values between Ku702-NLS/PEI and PEI significantly different (p≤0.043; n = 4). Statistical analysis was performed using the Mann-Whithney-U test.

Mentions: Using the IVIS in vivo imaging method it could be established that every type of gene vector complex was able to mediate gene transfer. Luciferin luminescence was measurable over all segments of the lungs of the tested groups of mice. In some contrast to the in vitro data, the Ku702-NLS/PEI/DNA mediated gene transfer was about 20% lower than PEI/DNA, but this effect was not statistically significant. s1Ku702-NLS/PEI/DNA mediated gene transfer was about 12% and s2Ku702/PEI/DNA mediated gene transfer about 28% higher compared to PEI/DNA (n.s.). In addition to the in vivo measurements with IVIS, lung homogenisates were analyzed for the presence of luciferase activity. In this analysis, the Ku702-NLS/PEI mediated gene transfer was about 46% higher, s1Ku702-NLS/PEI/DNA about 77% and s2Ku702/DNA about 9% higher compared to PEI/DNA. The values are significantly different from control (p≤0.043; n = 4) (Figure 4). Although the IVIS measurements may not have reflected the in vitro results, the analyses in lung homogenisates partly confirmed the in vitro data.


Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery.

Matschke J, Bohla A, Maucksch C, Mittal R, Rudolph C, Rosenecker J - PLoS ONE (2012)

Analysing luciferase expression in lung homogenisates after nasal instillation of gene transfer agents.1, Ku702-NLS/PEI/DNA; 2, s1Ku702-NLS/PEI/DNA; 3, s2Ku702/PEI/DNA; 4, PEI/DNA. Gene vector complexes were formulated as follows: 30 µg DNA; NLS: ± ratio = 5; PEI: N/P ratio = 10. Luciferase activity was measured in lung homogenisates 24 h after application of gene vectors. Values between Ku702-NLS/PEI and PEI significantly different (p≤0.043; n = 4). Statistical analysis was performed using the Mann-Whithney-U test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275586&req=5

pone-0024615-g004: Analysing luciferase expression in lung homogenisates after nasal instillation of gene transfer agents.1, Ku702-NLS/PEI/DNA; 2, s1Ku702-NLS/PEI/DNA; 3, s2Ku702/PEI/DNA; 4, PEI/DNA. Gene vector complexes were formulated as follows: 30 µg DNA; NLS: ± ratio = 5; PEI: N/P ratio = 10. Luciferase activity was measured in lung homogenisates 24 h after application of gene vectors. Values between Ku702-NLS/PEI and PEI significantly different (p≤0.043; n = 4). Statistical analysis was performed using the Mann-Whithney-U test.
Mentions: Using the IVIS in vivo imaging method it could be established that every type of gene vector complex was able to mediate gene transfer. Luciferin luminescence was measurable over all segments of the lungs of the tested groups of mice. In some contrast to the in vitro data, the Ku702-NLS/PEI/DNA mediated gene transfer was about 20% lower than PEI/DNA, but this effect was not statistically significant. s1Ku702-NLS/PEI/DNA mediated gene transfer was about 12% and s2Ku702/PEI/DNA mediated gene transfer about 28% higher compared to PEI/DNA (n.s.). In addition to the in vivo measurements with IVIS, lung homogenisates were analyzed for the presence of luciferase activity. In this analysis, the Ku702-NLS/PEI mediated gene transfer was about 46% higher, s1Ku702-NLS/PEI/DNA about 77% and s2Ku702/DNA about 9% higher compared to PEI/DNA. The values are significantly different from control (p≤0.043; n = 4) (Figure 4). Although the IVIS measurements may not have reflected the in vitro results, the analyses in lung homogenisates partly confirmed the in vitro data.

Bottom Line: We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method.The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo.This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Ludwig-Maximilians University, Munich, Germany.

ABSTRACT
Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

Show MeSH