Lipid-mediated siRNA delivery down-regulates exogenous gene expression in the mouse brain at picomolar levels.
BACKGROUND
Efficient in vivo vectors are needed to exploit the enormous potential of RNA interference (RNAi). Such methods require optimisation for specific delivery routes, tissues and usages. We tested the capacity of different non-viral vectors and formulation methods for inhibition of exogenous (luciferase) gene expression when used to introduce small interfering RNA (siRNA) into the mouse brain in vivo.


METHODS
Polyethylenimine (PEI)-based polyplexes and JetSI (a mixture of cationic lipids)-based lipoplexes were used to vectorise plasmid DNA encoding the firefly Photinus pyralis luciferase gene and picomolar amounts of siRNA directed against this gene. Two controls were used, DNA encoding an unrelated luciferase from Renilla reniformis and a mutated siRNA sequence.


RESULTS
First, we found that linear PEI, although efficient for delivering nucleic acids to cells, did not permit development of siRNA activity within the dose range tested (<0.5 pmol). Second, various combinations of cationic lipids were tried and the best formulation was found to be a combination of JetSI with the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE). Efficient inhibition of target, firefly luciferase was obtained with exceedingly low amounts of siRNA: 78 +/- 6% inhibition at 24 h post-transfection with 0.2 pmol siRNA. This inhibition was dose-dependent and specific. No effect was seen on the control gene, co-transfected Renilla luciferase, and the control mutated siRNA sequence had no effect on the targeted firefly luciferase.


CONCLUSIONS
We have optimised an efficient cationic lipoplex method for delivery of siRNA into the newborn mouse brain. Specific inhibition of exogenous target gene expression is obtained with picomolar amounts of siRNA.
Hassani
Zahra
Z
Laboratoire d'Evolution des Régulations Endocriniennes, MNHN, UMR CNRS 5166, 7 rue Cuvier 75231 Paris Cedex 5, France.
Lemkine
Gregory F
GF
Erbacher
Patrick
P
Palmier
Karima
K
Alfama
Gladys
G
Giovannangeli
Carine
C
Behr
Jean-Paul
JP
Demeneix
Barbara A
BA
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D005260:Female; D048888:Fireflies / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000378:metabolism*; D050356:Lipid Metabolism; D008156:Luciferases / Q000378:metabolism*; D008297:Male; D051379:Mice; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011094:Polyethyleneimine / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D048790:Renilla / Q000201:enzymology
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D010714:Phosphatidylethanolamines; D034741:RNA, Small Interfering; D011094:Polyethyleneimine; D008156:Luciferases