An NLS peptide covalently linked to linear DNA does not enhance transfection efficiency of cationic polymer based gene delivery systems.
BACKGROUND
Transfection with non-viral gene delivery vectors, such as cationic polymers, generally results in low transgene expression in vivo. This is likely due to poor cytoplasmic transport and intra-nuclear DNA delivery.


METHODS
In this study two strategies to improve nuclear import were investigated. Linear DNA constructs with or without an NLS peptide were prepared by PCR. Alternatively, linear DNA obtained by enzymatic cleavage followed by capping of both ends with DNA-hairpins was used. An NLS peptide was attached to one of the capped ends of the linear DNA. Both biodegradable (pDMAEAppz) and non-degradable polymers (PEI or pDMAEMA) were used to complex the DNA. Several cell types, dividing and non-dividing, were transfected with the linear DNA constructs containing a SV40-derived NLS peptide. Nuclear import of the DNA constructs was studied using digitonin-permeabilized cells.


RESULTS
Linear DNA prepared by PCR proved not useful as it was degraded from the 3'end. Linear DNA capped with hairpins was more successful with regard to stability. However, Cells transfected with linear DNA constructs by electroporation or by using cationic polymers with linear DNA containing a NLS peptide, failed to show significantly higher luciferase expression levels when compared to cells transfected with plasmid DNA or linear DNA without an NLS peptide attached. No nuclear localization was observed in digitonin-permeabilized cells.


CONCLUSIONS
Taken together, these data demonstrate that this nuclear localisation signal when attached to DNA is neither able to improve transfection efficiency of cationic polymers nor the nuclear import of the DNA constructs.
van der Aa
M A E M
MA
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB Utrecht, the Netherlands.
Koning
G A
GA
d'Oliveira
C
C
Oosting
R S
RS
Wilschut
K J
KJ
Hennink
W E
WE
Crommelin
D J A
DJ
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018274:Electroporation; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008689:Methacrylates / Q000378:metabolism; D008856:Microscopy, Fluorescence; D019913:Nuclear Localization Signals / Q000378:metabolism*; D009757:Nylons / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D008689:Methacrylates; D019913:Nuclear Localization Signals; D009757:Nylons; C407037:poly(2-(dimethylamino)ethyl methacrylate); D011094:Polyethyleneimine; D004247:DNA; D008156:Luciferases; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III