Citation

  • Authors: Bolcato-Bellemin, A. L., Bonnet, M. E., Creusat, G., Erbacher, P., Behr, J. P.
  • Year: 2007
  • Journal: Proc Natl Acad Sci U S A 104 16050-5
  • Applications: in vivo / DNA, STICKY SIRNA / in vivo-jetPEI

Method

30 µg of plasmid and 20 µg of siRNA, each in 50 µl of 5% glucose, were mixed separately with in vivo-jetPEI to a N/P of 8. The final volume of each complex was 100 µl. After 15 min incubation the plasmid complexes and siRNA complexes were mixed together. A total of 200 µl of DNA/siRNA mixture was injected retroorbitally into 8-week-old female mice.

Abstract

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery. We show here that making siRNAs "gene-like," via short complementary A(5-8)/T(5-8) 3' overhangs, increases complex stability, and hence RNase protection and gene silencing in vitro up to 10-fold. After decomplexation in the cytoplasm, sticky siRNA (ssiRNA) concatemers fall apart. ssiRNAs are therefore not inducing antiviral responses, as shown by the absence of IFN-beta production. Finally, transfection experiments in the mouse lung show that ssiRNA should be particularly suited to silencing with linear polyethylenimine in vivo.

Pubmed