The 16th International Symposium on Amyloidosis
タイトル；Design and Evaluation of shRNA Complex with Cyclodextrin/Dendrimer Conjugate for Treatment of Transthyretin Amyloidosis.
発表者；井上 雅理 氏
（熊本大学 大学院薬学教育部 製剤設計学分野）
INTRODUCTION: Hereditary amyloidogenic transthyretin (ATTR) amyloidosis is an autosomal dominant form of fatal hereditary amyloidosis characterized by systemic accumulation of ATTR amyloid fibrils in peripheral nerves and various organs. Previously, we reported that glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) inhibited ATTR amyloid fibril formation through hydrophobic interaction. In addition, we reported that GUG-β-CyD/Starburst® polyamidoamine dendrimer (Dendrimer) conjugate (GUG-β-CDE) has potential to exhibit superior gene transfer activity [1, 2]. In this study, we newly prepared GUG-β-CDE/TTR short hairpin RNA (shTTR) complex, and evaluated the RNAi effect against TTR gene expression, inhibitory effects on the formation of ATTR amyloid fibril, and dissolution effects on ATTR amyloid fibril in vitro and in vivo.
MATERIALS & METHODS: GUG-β-CDE having the degree of substitution of GUG-β-CyD of 1.8 was prepared. The secondary structures of TTR V30M in solution were examined by circular dichroism (CD) spectroscopy. Thioflavin-T (Th-T) assay was performed for the quantitation of TTR V30M amyloid fibril. Human TTR V30M Transgenic (Tg) rats (9 months) were used as a rats’ model of early onset TTR V30M amyloidosis, in which TTR started deposition in colon, and (15 months) as a rats’ model of late onset TTR V30M amyloidosis a model, in which TTR was already deposited in colon. Human TTR V30M Tg rats (9 and 15 months old) were treated with GUG-β-CDE (2 mg/kg)/shTTR (0.053 mg/kg) complex dissolved in 5% mannitol solution (500 μL) through tail vain injection twice per week during 3 months. TTR deposition in colon of Tg rats were evaluated by immunostaining.
RESULTS: GUG-β-CDE/shTTR complex greatly reduced the structural transitions of TTR V30M towards the β-sheet rich structure, and significantly inhibited TTR V30M amyloid fibril formation. In addition, GUG-β-CDE/shTTR complex drastically dissolved TTR V30M amyloid fibril. The RNAi effect of GUG-β-CDE/shTTR complex was higher than that of Dendrimer/shTTR complex in human hepatocyte HepG2 cells. Moreover, in rats’ model of early and late onset TTR V30M amyloidosis, intravenous administration of GUG-β-CDE/shTTR complex to TTR V30M Tg rats significantly suppressed TTR mRNA level in liver and TTR deposition in colon.
DISCUSSION & CONCLUSIONS: GUG-β-CDE/shTTR complex may have potential as a novel therapeutic agent for ATTR amyloidosis through the multiple function, such as RNAi effects, inhibitory effects on ATTR amyloid fibril formation, and dissolution ability for the ATTR amyloid fibrils.
REFERENCES: 1. Anno T. et al. Potential use of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate (G2) as a DNA carrier in vitro and in vivo. J. Drug Target 2012;20:272-280. 2 Anno T. et al. Possible enhancing mechanisms for gene transfer activity of glucuronylglucosyl-β-cyclodextrin/dendrimer conjugate. Int. J. Pharm. 2012;426:239-247.