The systemic administration of an anti-miRNA oligonucleotide encapsulated pH-sensitive liposome results in reduced level of hepatic microRNA-122 in mice

Abstract

Efficient delivery continues to be a challenge in microRNA (miRNA) therapeutics. We utilized a pH-sensitive multifunctional envelope-type nano device (MEND) containing a pH-sensitive lipid YSK05 (YSK05-MEND) to regulate liver specific miRNA-122 (miR-122). Anti-microRNA oligonucleotides including 2′-OMe and phosphorothioate modifications against miR-122 (AMO122) were encapsulated in the YSK05-MEND. Despite the lower uptake, the YSK05-MEND showed a higher activity in liver cancer cells than Lipofectamine2000 (LFN2k) due to efficient endosomal escape. Cytotoxicity was minimal at 100 nM of AMO122 in YSK05-MEND treated cells, but LFN2k showed toxicity at 50 nM. When mice were administrated with free AMO122, it was eliminated via the kidney due to its molecular weight, and lesser amounts were detected in the liver. Conversely, the YSK05-MEND delivered higher amounts of the AMO122 to the liver. Systemic administration of YSK05-MEND induced the knockdown of miR-122 and an increase in target genes in the liver, and a subsequent reduction in plasma cholesterol at a dose of 1 mg AMO/kg while free AMO122 showed no activity at the same dose. The effect of AMO122 delivered by YSK05-MEND persisted for over 2 weeks. These results suggest that YSK05-MEND is a promising system for delivering AMOs to the liver.

Introduction

MicroRNAs (miRNAs) are a class of small non-coding RNAs (~ 22 nt) that regulate gene expression by binding to the 3′-untranslated region (3′-UTR) of target genes, triggering the degradation of messenger RNA (mRNA) or the inhibition of protein translation [1]. MicroRNA-122 (miR-122) is a conserved liver-specific miRNA that accounts for 70% of the total miRNA population [2] and plays important roles in liver physiology, such as lipid metabolism [3], [4], diseases in hepatic virus C (HCV) infections [5] and hepatocellular carcinoma (HCC) [6]. In addition, the expression of miR-122 in other organs such as heart is negligible [7]. Thus, miR-122 is an attractive and selective therapeutic target for the treatment of liver diseases [8].

A number of attempts have been made to induce the specific inhibition of endogenous miRNAs in vivo. Plasmid DNA vectors that express miRNA sponges, which contain multiple tandem miRNA binding sites, have been designed to competitively inhibit miRNA functions in mammalian cells [9]. It was recently reported that the intravenous administration of recombinant adeno associated virus (rAAV) vectors with miRNA tough decoys (TuDs) result in reduced levels of miR-122 and serum cholesterol [10]. Anti-microRNA oligonucleotides (anti-miRs) have been widely employed to inhibit miRNAs, including a variety of nucleoside modifications, such as 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and locked nucleic acid (LNA) was developed in an attempt to enhance binding affinity to target miRNAs with phosphorothioate (PS) linkages to improve nuclease resistance [11]. The systemic injection of these anti-miRs against miR-122 (anti-miR122) into mice resulted in a reduction in cholesterol levels [3], [4], [12], [13], [14]. Miravirsen, an LNA-modified anti-miR122, is currently in phase 2 clinical trials for the treatment of HCV [15].

However, large doses of anti-miR-122 are required to induce the phenotype when the free form of anti-miR-122 is injected due to renal excretion and poor tissue selectivity as well as cellular uptake [16], [17], [18]. To overcome these issues, systems that are capable of delivering nucleic acids to a targeted organ are desired [18]. Lipid based nanoparticles have been extensively investigated as vehicles for delivering siRNAs, miRNAs, as well as anti-miRs [19], [20], [21], [22], [23]. We developed a multifunctional envelope-type nano device (MEND), in which nucleic acids are encapsulated within a lipid envelope [24], [25]. We recently synthesized a pH-sensitive cationic lipid, referred to as YSK05 [26]. A MEND composed of YSK05 (YSK05-MEND) showed efficient pH-sensitive fusogenic properties and a higher gene knockdown ability than a commercially available transfection reagent, Lipofectamine 2000 (LFN2k) in HeLa cells [26]. The systemic administration of YSK05-MEND modified with PEG delivered siRNA to tumor tissue in renal cell carcinoma xenograft mice via the enhanced permeability and retention (EPR) effect and induced the knockdown of approximately 50% of the target gene at a dose of 3 mg/kg body weigh [27]. These findings indicate that YAK05-MEND has the potential for use in nucleic acid delivery both in vitro and in vivo.

In the present study, we report on an investigation of whether the YSK05-MEND could be applicable for use in an anti-miR-122 delivery system to murine liver. We first evaluated the physical properties of the YSK05-MEND encapsulating anti-miR-122 modified with 2′-OMe and PS linkages (AMO122) and its activity in murine hepatoma cells in comparison with LFN2k. For in vivo studies, we compared the activity of systemically administrated YSK05-MEND with free AMO in terms of distribution in the liver and kidney, and the effect of antagonism of target miR-122 on the increase in the expression level of genes that are regulated by miR-122 and the subsequent reduction in plasma cholesterol levels. The findings indicate that the YSK05-MEND has the potential for use in the efficient delivery of AMOs to murine liver.