Highly efficient silencing of microRNA by heteroduplex oligonucleotides

Kotaro Yoshioka,Yutaro Asami,Takanori Yokota,Kazutaka Nishina,Taiki Kunieda,Kie Yoshida-Tanaka,Hiroya Kuwahara,Tetsuya Nagata,Takeshi Wada,Yumiko Sujino,Satoshi Obika,Rintaro Iwata Hara,Haruka Miyata,Huijia Guo,Wenying Piao

doi:10.1093/nar/gkz492

Abstract

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.

Highlights

MicroRNAs are endogenous small non-coding RNAs (21–23 nucleotides) that inhibit messenger RNA (mRNA) posttranscriptionally [1] and play crucial roles in various physiological processes and responses to pathogens [2]
Dose-response curve analyses (Figure 1D) revealed dose dependent miRNA-binding by the parent antimiR and heteroduplex oligonucleotide (HDO)-antimiR, and showed 12-fold greater binding efficiency by HDO-antimiR compared to the parent antimiR (50% binding-dose by HDO-antimiR or the parent antimiR: 1.3 versus 15 nmol/kg respectively)
This finding is surprising because previous studies demonstrated that HDO targeting ‘messenger RNA’ without a ligandconjugation to complementary strand cannot improve the potency of mRNA silencing [31,40]

Summary

Introduction

MicroRNAs (miRNAs) are endogenous small non-coding RNAs (21–23 nucleotides) that inhibit mRNA posttranscriptionally [1] and play crucial roles in various physiological processes and responses to pathogens [2]. Steric-blocking and degradation of target RNA are major silencing mechanisms of ASO including antimiR [7,10,11]. RNA-degradation mechanism has potential advantages over steric-blocking. While stericblocking type of antimiRs bind and sequester single target miRNAs in RNA induced silencing complexes (RISC) and are not subsequently recycled, degradation-type of antimiRs can bind and be released from target miRNAs multiple times, allowing greater turnover ratios [7,8,14]. Degradation-type of antimiRs have lower cell toxicity, because degradation of the targeted miRNA allows RISC to be recycled and remain functional. Duplexes of steric-blocking type of antimiR and target miRNAs may occupy RISC, causing cell toxicity by interfering with the maturation of other miRNAs [10]

Methods

Results

Conclusion