研究発表を行った学会；The XVIth International Symposium on Amyloidosis (ISA2018)
タイトル； Therapeutic Approach for Transthyretin Amyloidosis by Targeted Delivery of siRNA Ternary Complex with Cyclodextrin/dendrimer Conjugate.
（熊本大学 医学部附属病院 薬剤部・臨床薬物動態分野）
INTRODUCTION: RNA interference (RNAi) therapy has tremendous potential for treating various intractable diseases by suppressing expression of target genes. Lack of safe and efficacious delivery system poses a major challenge limiting clinical advancement of RNAi therapeutics. In particular, with regard to treatment of transthyretin-related familial amyloidotic polyneuropathy (TTR-FAP), which is a progressive and fatal hereditary amyloidosis, hepatocyte-targeted delivery is desired because amyloidogenic TTR is predominantly produced by hepatocyte. Recently, we have reported that the polyamidoamine dendrimer conjugate with α-cyclodextrin bearing lactose (Lac-α-CDE) provided remarkable aspects as hepatocyte-targeted siRNA carrier in vitro and in vivo. Lac-α-CDE binary complex with TTR siRNA (siTTR) had the potent RNAi effect against TTR expression through selective cellular uptake mediated by asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes cell surface, efficient endosome escape and negligible cytotoxicity. However, Lac-α-CDE/siTTR binary complex does not have enough serum stability and in vivo transfection efficacy to exhibit its therapeutic effect. In the present study, to improve the serum stability, hepatocyte accumulation, and in vivo RNAi effect of Lac-α-CDE/siRNA binary complex, we developed a novel hepatocyte-targeted siRNA delivery carrier consisting of Sacran (Lac-α-CDE/siRNA/sacran ternary complex), and evaluated its therapeutic effects on TTR amyloidosis.
MATERIALS & METHODS: We newly developed the ternary complexes consisting of Lac-α-CDE, siRNA and the anionic polysaccharide sacrans (Lac-α-CDE/siTTR/sacran ternary complex), and evaluated their utility as siRNA transfer carriers. Three kinds of the low-molecular-weight sacrans, i.e. sacran (100) (Mw 44,889 Da), sacran (1,000) (Mw 943,692 Da) and sacran (10,000) (Mw 1,488,281 Da) were used. Human TTR and mouse TTR siRNAs were kindly provided by Alnylam Pharmaceuticals, Inc. We evaluated uptake and RNAi effect of siTTR ternary complexes with Lac-α-CDE/sacran in vitro and in vivo.
RESULTS: Lac-α-CDE/siRNA/sacran ternary complexes were prepared by adding the low-molecular-weight sacrans to the Lac-α-CDE/siRNA binary complex solution. Cellular uptake of the ternary complex with sacran (100) was higher than that of the binary complex or the other ternary complexes with sacran (1,000) and sacran (10,000) in HepG2 cells. Lac-α-CDE/siTTR/sacran (100) ternary complex showed the significant RNAi effect in human hepatocyte HepG2 cells, resulting from suitable physicochemical properties, ASGPR-mediated cellular uptake, serum stability and the localization of the siRNA in cytoplasm without cytotoxicity. High levels of siRNA and Lac-α-CDE in liver were observed after intravenous administration of the ternary complex rather than that of the binary complex. Moreover, intravenous administration of the ternary complex induced the significant RNAi effect in the liver of mice with negligible change of blood chemistry values.
DISCUSSION & CONCLUSIONS: Our findings provided that Lac-α-CDE/siRNA/sacran (100) ternary complex elicits the potent RNAi effects through high ASGPR-mediated cellular uptake, serum stability and localization in cytoplasm without cytotoxicity. Importantly, the significant RNAi effects and the negligible side effects were observed even after intravenous administration of Lac-α-CDE/siRNA/sacran (100) ternary complex into mice due to increase in the complex stability and accumulation of carrier and siRNA in liver. Taken together, these results suggest that the Lac-α-CDE/siRNA/sacran (100) ternary complex has the potential as a novel hepatocyte-targeted siRNA delivery system in vitro and in vivo. Therefore, Lac-α-CDE/siRNA/sacran (100) complex has utility as a novel therapeutic approach for TTR amyloidosis.