Cationic liposome-mediated delivery of siRNAs in adult mice

https://doi.org/10.1016/j.bbrc.2003.11.057Get rights and content

Abstract

RNA interference mediated by small interfering RNAs (siRNAs) is a powerful tool for dissecting gene function and drug target validation. siRNAs can be synthesized in large quantities and thus can be used to analyze a large number of sequences emerging from genome projects in a cost-effective manner. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. We used a fluorescein-labeled siRNA to investigate cationic liposome-mediated intravenous and intraperitoneal delivery in adult mice. We show that this simple approach can deliver siRNAs into various cell types. In addition, we show that in contrast to mouse cells, siRNAs can activate the non-specific pathway in human freshly isolated monocytes, resulting in TNF-α and IL-6 production. Taken together, the data provide a basis for lipid-mediated systemic delivery of siRNAs and indicate that certain siRNA sequences can activate the innate immunity response genes that can be beneficial for the treatment of cancer.

Section snippets

Materials and methods

Reagents. 1,2 Dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from Roche Diagnostics. FITC-labeled siRNAs were chemically synthesized and purified by Eurogentec. The siRNAs were annealed in transfection buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) at 3 μg/μl.

Preparation of siRNA–liposome complexes for i.v. injection of mice. For each mice 20–100 μg of siRNAs in 60 μl of transfection buffer was transferred into a sterile Eppendorf tube. In a separate sterile polystyrene tube 50 μg of DOTAP was

Cationic liposome-mediated intravenous nucleic acid delivery

Similar to antisense oligonucleotides and ribozymes, synthetic siRNAs are generally delivered to cells via liposome-based transfection reagents [6]. These reagents offer the possibility of developing pharmaceutical compounds for local or systemic delivery. Notably, most patients who die from cancer have subclinical metastatic disease present at the time of diagnosis [9]. Thus, it is essential that molecular medicine-based therapies to treat cancer be adjusted to a systemic administration. In

Discussion

The study of nucleic acids has revealed remarkable properties of RNA molecules that could make them attractive therapeutic agents, independent of their well-known ability to encode biologically active proteins [13]. This application is now mainly driven by small interfering RNAs that serve as guide sequences to induce target-specific mRNA cleavage via the activation of a highly conserved regulatory mechanism, called RNA interference or posttranscriptional gene silencing [3]. However, crucial

Acknowledgements

This work was supported by the Norwegian Cancer Society. We thank Dr. Anne Dybwad for critical reading of the manuscript and Khu Ky Cuong for excellent technical assistance.

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