Kiyomi Sasaki

Cholesterol-lowering Action of BNA-based Antisense Oligonucleotides Targeting PCSK9 in Atherogenic Diet-induced Hypercholesterolemic Mice

Recent findings in molecular biology implicate the involvement of proprotein convertase subtilisin/kexin type 9 (PCSK9) in low-density lipoprotein receptor (LDLR) protein regulation. The cholesterol-lowering potential of anti-PCSK9 antisense oligonucleotides (AONs) modified with bridged nucleic acids (BNA-AONs) including 2′,4′-BNA (also called as locked nucleic acid (LNA)) and 2′,4′-BNANC chemistries were demonstrated both in vitro and in vivo. An in vitro transfection study revealed that all of the BNA-AONs induce dose-dependent reductions in PCSK9 messenger RNA (mRNA) levels concomitantly with increases in LDLR protein levels. BNA-AONs were administered to atherogenic diet-fed C57BL/6J mice twice weekly for 6 weeks; 2′,4′-BNA-AON that targeted murine PCSK9 induced a dose-dependent reduction in hepatic PCSK9 mRNA and LDL cholesterol (LDL-C); the 43% reduction of serum LDL-C was achieved at a dose of 20 mg/kg/injection with only moderate increases in toxicological indicators. In addition, the serum high-density lipoprotein cholesterol (HDL-C) levels increased. These results support antisense inhibition of PCSK9 as a potential therapeutic approach. When compared with 2′,4′-BNA-AON, 2′,4′-BNANC-AON showed an earlier LDL-C–lowering effect and was more tolerable in mice. Our results validate the optimization of 2′,4′-BNANC-based anti-PCSK9 antisense molecules to produce a promising therapeutic agent for the treatment of hypercholesterolemia.

Thermodynamic and Kinetic Effects of Morpholino Modification on Pyrimidine Motif Triplex Nucleic Acid Formation under Physiological Condition

Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.

Interrupted 2′-O,4′-C-Aminomethylene Bridged Nucleic Acid Modification Enhances Pyrimidine Motif Triplex-Forming Ability and Nuclease Resistance Under Physiological Condition

Due to instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex formation-based strategies in vivo, such as regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis. To this end, we investigated the effect of our previously reported chemical modification, 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′- BNANC) modification, introduced into interrupted and continuous positions of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at physiological pH. The interrupted 2′,4′-BNANC modifications of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold, and significantly increased the nuclease resistance of TFO. On the other hand, the continuous 2′,4′-BNANC modification of TFO showed lower ability to promote the triplex formation at physiological pH than the interrupted 2′,4′-BNANC modifications of TFO, and did not significantly change the nuclease resistance of TFO. Selection of the interruptedly 2′,4′-BNANC-modified positions in TFO was more favorable for achieving the higher binding affinity of the pyrimidine motif triplex formation at physiological pH and the higher nuclease resistance of TFO than that of the continuously 2′,4′-BNANC-modified positions in TFO. We conclude that the interrupted 2′,4′-BNANC modification of TFO could be a key chemical modification to enhance pyrimidine motif triplex-forming ability and nuclease resistance under physiological condition, and may eventually lead to progress in various triplex formation-based strategies in vivo.

DX DNAs as Templates for Multiple Arrangement of Zinc Fingers

The present paper reveals that double crossover-DNAs (DX) serve as scaffolds for the multiple arrangement of a [Ru(bpy)(3)](2+)-bound zinc finger (ZF) protein, Ru-ZF. This series of results would lead to the realization of the two-dimensional arrangement of functional molecules and nanomaterials on DX-tiles.