Noriko Tarashima

Chemistry, Properties, and in Vitro and in Vivo Applications of 2′‐O‐Methoxyethyl‐4′‐thioRNA, a Novel Hybrid Type of Chemically Modified RNA

We report the synthesis, properties, and in vitro and in vivo applications of 2′‐O‐methoxyethyl‐4′‐thioRNA (MOE‐SRNA), a novel type of hybrid chemically modified RNA. In its hybridization with complementary RNA, MOE‐SRNA showed a moderate improvement of Tm value (+3.4 °C relative to an RNA:RNA duplex). However, the results of a comprehensive comparison of the nuclease stability of MOE‐SRNA relative to 2′‐O‐methoxyethylRNA (MOERNA), 2′‐O‐methyl‐4′‐thioRNA (Me‐SRNA), 2′‐O‐methylRNA (MeRNA), 4′‐thioRNA (SRNA), and natural RNA revealed that MOE‐SRNA had the highest stability (t1/2>48 h in human plasma). Because of the favorable properties of MOE‐SRNA, we evaluated its in vitro and in vivo potencies as an anti‐microRNA oligonucleotide against miR‐21. Although the in vitro potency of MOE‐SRNA was moderate, its in vivo potency was significant for the suppression of tumor growth (similar to that of MOERNA).

Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4′-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4′-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4′-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response.

The novel functional nucleic acid iRed effectively regulates target genes following cytoplasmic delivery by faint electric treatment

Abstract An intelligent shRNA expression device (iRed) contains the minimum essential components needed for shRNA production in cells, and could be a novel tool to regulate target genes. However, general delivery carriers consisting of cationic polymers/lipids could impede function of a newly generated shRNA via electrostatic interaction in the cytoplasm. Recently, we found that faint electric treatment (fET) of cells enhanced delivery of siRNA and functional nucleic acids into the cytoplasm in the absence of delivery carriers. Here, we examined fET of cells stably expressing luciferase in the presence of iRed encoding anti-luciferase shRNA. Transfection of lipofectamine 2000 (LFN)/iRed lipoplexes showed an RNAi effect, but fET-mediated iRed transfection did not, likely because of the endosomal localization of iRed after delivery. However, fET in the presence of lysosomotropic agent chloroquine significantly improved the RNAi effect of iRed/fET to levels that were higher than those for the LFN/iRed lipoplexes. Furthermore, the amount of lipid droplets in adipocytes significantly decreased following fET with iRed against resistin in the presence of chloroquine. Thus, iRed could be a useful tool to regulate target genes following fET-mediated cytoplasmic delivery with endosomal escape devices.