830. Efficient Transfer of Synthetic Small Interfering RNAs through Atelocollagen-Mediated Delivery System
Authors: Shunji Nagahara, Yoshiko Minakuchi, Fumitaka Takeshita, Kimi Honma, Koji Hanai, Akihiko Sano, Takahiro Ochiya
Journal: Molecular Therapy (2004)

Since it was observed that RNA interference which is an autologous protecting and regulating mechanism of eukaryotes can be worked artificially by introducing synthetic small interfering RNAs (siRNA), silencing of gene expression by siRNA is rapidly becoming one of the most powerful tool for genetic analysis of a wide variety of mammalian cells. However, with the progress of the studies, it becomes well known that silencing of gene expression using siRNA is transient, and siRNA shows very low efficiency in vivo. Although to overcome this problem various methods such as the use of plasmid DNAs, and viral vectors encoding siRNA and the use of liposomes have been investigated, no sufficient method for clinical use has been established. Recently, atelocollagen-mediated delivery system has been established for therapeutic use of nucleic acid medicines (NAMs) such as plasmid DNA and antisense DNA. Atelocollagen has been widely used because of its high biocompatibility derived from its low toxicity, low immunogenicity and biodegradability. Previous studies have shown that this delivery system enhances the therapeutic effects of the NAMs and prolongs their period of potent action. In this study, we demonstrated that the delivery system enhances and prolongs silencing effect of siRNA in vitro and in vivo. SiRNAs (21mer) designed to inhibit expression of HST-1/FGF-4 gene (HST-siRNA) or luciferase gene (LUC-siRNA) were synthesized. The siRNA/atelocollagen complex was formed by mixing siRNAs with atelocollagen, and these complexes were prepared in an injectable form and can be administered by conventional methods. When the LUC-siRNA formed complex with atelocollagen, and the complex was administrated to HeLa cells which continuously expressed luciferase gene, its inhibitory effect was dramatically enhanced, and the effect revealed about two folds more than the ordinary polycation transfer method. Human testicular tumor cells, NEC8, of which cell growth is specifically inhibited by suppression of HST-1/FGF-4 gene was employed to determine whether complex formation could prolong action of HST-siRNAs. The NEC8 cells bearing the HST-siRNA with polycation reagent showed an inhibition of cell growth for only 3 days. In contrast, the siRNA/atelocollagen complex allowed inhibition of cell growth for a longer period of time. Noninvasive in vivo biophotonic imaging analysis was used to detect long term gene silencing in mice engrafted with melanoma cells expressing luciferase gene. Gene suppression efficiency of the LUC-siRNA/atellocollagen complex administrated into mice was superior to that of conventional siRNA administration methods. Furthermore, efficient growth inhibition of the siRNA/atelocollagen complex to NEC8 cells grafted orthotopically was also observed. In summary, atelocollagen-mediated NAMs delivery system holds great potential in significantly advancing the practical application of gene suppression using siRNAs for functional genomics and therapeutics.