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Efficient RNA Interference Delivery via Simian Virus 40 Vectors Packaged In Vitro
SV40 vectors packaged in vitro (SV40-IVPs, also called SV40 pseudovirions) are known to be efficient delivery systems for supercoiled DNA plasmids. SV40-IVPs can be used to efficiently transfer DNA up to 17.7 kb in size into a variety of mammalian cells both in vitro and in vivo. They are probably safer than the recombinant SV40 delivery system (Kimchi-Sarfaty C et al. Biotechniques 37: 270–5, 2004) or any other viral delivery system (Gottesman MM. Cancer Gene Ther 10: 501–8, 2003), because no packaging cell line or viral DNA is present. We demonstrated that the packaging and highly efficient delivery of siRNAs into two human cell lines, HeLa and human lymphoblastoids, in vitro can also be accomplished using SV40-IVPs. Our results were confirmed by way of a complete knockout of green fluorescent protein (GFP) expression. RNAi is mediated through the RNA-induced silencing complex, which has single-stranded RNA species 21 to 23 nucleotides long. This complex recognizes target RNAs and cleaves them in a position-dependent manner. Since some of this activity takes place within the cytoplasm, SV40-IVPs may serve as good carriers for such an agent. We used SV40-IVPs to deliver both plasmids expressing short hairpin RNAs (shRNAs) and synthetic siRNAs into human lymphoblastoid cells in suspension and HeLa adherent cells. Although delivery of synthetic siRNAs to adherent cells has previously been accomplished via polyplex delivery systems and cationic lipids, these methods do not work well on cells in suspension. Initially, we showed that SV40-IVPs can deliver shRNAs that interfere with GFP expression (SV40-IVP-shGFPs). Human lymphoblastoid cells that were transduced with a half reaction of SV40-IVP-shGFPs and a half reaction of SV40-IVP-GFPs did not show GFP expression (as transduced mock cells), in comparison with cells transduced with SV40-IVP-GFP only. We later demonstrated, by fluorescence-activated cell-sorting (FACS) and by confocal microscopy, a highly fluorescent signal in cells transduced by SV40-IVPs carrying fluorescence-tagged siRNA (SV40-IVP-siRNA.3.Fl), showing that siRNA can in fact be packaged and delivered by pseudovirions. A visible inhibition of GFP expression in HeLa cells, which continuously express GFP (under the influence of selective agent G418), was achieved by transducing cells with GFP-targeted siRNA (SV40-IVP-siGFP). A dose response of the packaged siGFP revealed that 1 μg of siGFP was sufficient to silence the GFP in 5 × 105 cells if it was packaged by SV40-IVPs. Finally, we compared the SV40-IVP delivery method to a lipid-transfection system using the HeLa cells that express high levels of GFP (Figure 1). The majority of the cells transduced with SV40-IVP-siRNA (SV40-IVP-siGFP) ceased GFP expression (approximately a 2 log shift), while lipid-transfected siRNA cells showed only a minor shift from the high levels of GFP observed in the control. The inhibition lasted longer using the pseudovirions, up to 6 days after transduction. Figure 1. Expression of small interfering RNA (siRNA) against HeLa cells stably expressing green fluorescent protein (GFP), with the siRNA delivered by simian virus 40 vectors packaged in vitro (SV40-IVP-siGFPs) or by lipid transduction. The majority of SV40-IVP-siGFP cells ceased GFP production (green), while most of the cells transfected with siRNAs (siGFPs) via lipids showed much higher expression of GFP (blue). Cells were analyzed 3 days after transduction. SV40-IVPs have several advantages over other delivery methods: They do not carry viral DNA, and therefore, there is no possibility of contamination. They are capable of transducing both non-dividing cells and cells in suspension, and they carry longer DNA fragments than other delivery methods. In this study, we showed that SV40-IVPs can be used for RNAi with very high efficiency and for a relatively long period of time.
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NA interference (RNAi) is the process of introducing small interfering RNA molecules (siRNAs) into eukaryotic cells to downregulate gene expression. There are two methods of introducing siRNAs into the cytoplasm: One is by using molecules like liposomes that are impregnated with double-stranded RNAs, and the other involves the use of viral vectors, in which the siRNAs are precursor RNAs expressed by the vectors (Huppi K et al. Mol Cell 17: 1–10, 2005). In our study, we propose a new delivery system, involving simian virus 40 (SV40) vectors, that can be incorporated into both methods. 