Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery.
BACKGROUND
One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell-permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS).


METHODS
Two CPP-NLS peptides which differ in the location of the NLS relative to the CPP were used: S4 13-PV and PV-S4 13. The peptides were attached to pDNA using electrostatic interactions. Gel-electrophoresis and fluorescent assays were performed to evaluate pDNA-peptide interactions and condensation effects. Confocal microscopy was used to evaluate pDNA-peptide interaction inside cells. Transfection studies were conducted with the luciferase gene, using pDNA-peptides alone, or with the application of TUS.


RESULTS
Attachment of both peptides to pDNA condensed the pDNA, with higher affinity for the S4(13)-PV peptide. This interaction protected pDNA from endonucleases, but was also reversible. Both peptides mediated pDNA delivery to cell cytoplasm, but less significantly to the nucleus. Thus, both peptides produced transfection in cells, when added after incubation with DNA, with higher transfection-level for PV-S4 13. Application of TUS increased transfection mediated by these peptides, but was not higher compared to transfection using TUS and pDNA alone.


CONCLUSIONS
This study suggests that CPP-NLS peptides may be used for condensing pDNA and bringing it into the cell cytoplasm, but their ability to mediate nuclear import of pDNA is insignificant.
Duvshani-Eshet
Maayan
M
The Faculty of Biotechnology and Food Engineering, The Technion-Israel institute of Technology, Haifa Israel.
Keren
Hadas
H
Oz
Shira
S
Radzishevsky
Inna S
IS
Mor
Amram
A
Machluf
Marcelle
M
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000737:chemistry; D019913:Nuclear Localization Signals / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D014162:Transfection / Q000379:methods*; D014465:Ultrasonics
D019913:Nuclear Localization Signals; D010455:Peptides; D004247:DNA