Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo.
BACKGROUND
To develop more efficient non-viral vectors, we have previously described a novel approach to attach a nuclear localisation signal (NLS) to plasmid DNA, by generating a fusion protein between the tetracycline repressor protein TetR and an SV40 NLS peptide (TetR-NLS). The high affinity of TetR for the DNA sequence tetO is used to bind the NLS to DNA. We have now investigated the ability of this system displaying the SV40 NLS or HIV-1 TAT peptide to enhance nuclear import of a minimised DNA construct more suitable for in vivo gene delivery: a minicircle.


METHODS
We have produced a new LacZ minicircle compatible with the TetR system. After transfection of the minicircle in combination with TetR-NLS or TetR-TAT using different transfection agents, we first measured beta-galactosidase activity in vitro. We then used a special delivery technique, in which DOTAP/cholesterol liposomes and DNA/protein complexes are sequentially injected intravenously, to evaluate the activity of this system in vivo.


RESULTS
In vitro results showed a 30-fold increase in transfection efficiency of the nuclear-targeted minicircle compared to normal plasmid lipofection. Results on cell cycle arrested cells seem to indicate a different mechanism between the TetR-NLS and TetR-TAT. Finally, we demonstrate a more than 6-fold increase in beta-galactosidase expression in the mouse lung using the minicircle and the TetR-TAT protein. This increase is specific for the peptide sequence and is not observed with the control protein TetR.


CONCLUSIONS
Our results indicate that the combination of a minicircle DNA construct with a TetR nuclear-targeting system is able to potentiate gene expression of non-viral vectors.
Gene Therapy Research Group, Division of Biomedical Science, Imperial College London, London SW7 2AZ, UK.
Laurence
L
Vaysse
Lisa G
LG
Gregory
Richard P
RP
Harbottle
Eric
E
Perouzel
Oleg
O
Tolmachov
Charles
C
Coutelle
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004270:DNA, Circular / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D041681:NIH 3T3 Cells; D013752:Tetracycline / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes; D001616:beta-Galactosidase / Q000378:metabolism
D004270:DNA, Circular; D001616:beta-Galactosidase; D013752:Tetracycline