Takayuki Mizutani

Systemically Injected Exosomes Targeted to EGFR Deliver Antitumor MicroRNA to Breast Cancer Cells

Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2-/- mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.Despite the therapeutic potential of nucleic acid drugs, their clinical application has been limited in part by a lack of appropriate delivery systems. Exosomes or microvesicles are small endosomally derived vesicles that are secreted by a variety of cell types and tissues. Here, we show that exosomes can efficiently deliver microRNA (miRNA) to epidermal growth factor receptor (EGFR)-expressing breast cancer cells. Targeting was achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the GE11 peptide. Intravenously injected exosomes delivered let-7a miRNA to EGFR-expressing xenograft breast cancer tissue in RAG2(-/-) mice. Our results suggest that exosomes can be used therapeutically to target EGFR-expressing cancerous tissues with nucleic acid drugs.

MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC.

Efficacy of a novel class of RNA interference therapeutic agents.

RNA interference (RNAi) is being widely used in functional gene research and is an important tool for drug discovery. However, canonical double-stranded short interfering RNAs are unstable and induce undesirable adverse effects, and thus there is no currently RNAi-based therapy in the clinic. We have developed a novel class of RNAi agents, and evaluated their effectiveness in vitro and in mouse models of acute lung injury (ALI) and pulmonary fibrosis. The novel class of RNAi agents (nkRNA®, PnkRNA™) were synthesized on solid phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a central stem and two loops. They are resistant to degradation and suppress their target genes. nkRNA and PnkRNA directed against TGF-β1mRNA ameliorate outcomes and induce no off-target effects in three animal models of lung disease. The results of this study support the pathological relevance of TGF-β1 in lung diseases, and suggest the potential usefulness of these novel RNAi agents for therapeutic application.

A novel platform to enable inhaled naked RNAi medicine for lung cancer

Small interfering RNA (siRNA)-based therapeutics have been used in humans and offer distinct advantages over traditional therapies. However, previous investigations have shown that there are several technical obstacles that need to be overcome before routine clinical applications are used. Currently, we are launching a novel class of RNAi therapeutic agents (PnkRNA™, nkRNA) that show high resistance to degradation and are less immunogenic, less cytotoxic, and capable of efficient intracellular delivery. Here, we develop a novel platform to promote naked RNAi approaches administered through inhalation without sophisticated delivery technology in mice. Furthermore, a naked and unmodified novel RNAi agent, such as ribophorin II (RPN2-PnkRNA), which has been selected as a therapeutic target for lung cancer, resulted in efficient inhibition of tumor growth without any significant toxicity. Thus, this new technology using aerosol delivery could represent a safe, potentially RNAi-based strategy for clinical applications in lung cancer treatment without delivery vehicles.

Screening of cell death genes with a mammalian genome-wide RNAi library

We report the construction and application of a mammalian genome-wide RNAi library. The oligodeoxynucleotides encoding approximately 200,000 shRNA sequences that targeted 47,400 human transcripts were inserted into a lentivirus vector pFIV-H1-puro, and a pool of pseudovirus particles with a complexity of approximately 200,000 were used to infect target cells. From the cells surviving apoptogenic Fas stimulation, four candidate shRNA sequences were obtained that provided resistance to Fas-induced cell death, including two shRNAs for caspase-8, an shRNA for Bid, and an shRNA for Fas. The reconstructed shRNAs with these sequences were shown to reduce expression of the respective gene products and increase survival after Fas stimulation. When similar selection was performed for tunicamycin-induced apoptosis, no shRNA strongly inhibiting tunicamycin-induced cell death was isolated, although a few reconstructed shRNAs led to a slight increase of survival. Thus, this genome-wide shRNA library proved useful for selection of genes that are involved in cell death, but some limitation was also revealed.

A useful method of identifying of miRNAs which can down-regulate Zeb-2

BackgroundAlthough identification of the target mRNAs of micro RNAs (miRNAs) is essential to understanding their function, the low complementarity between miRNAs and their target mRNAs has complicated this process. In this study, we sought to identify miRNAs which reduce the expression of the transcription factor Zeb-2, a transcriptional repressor of E-cadherin which is known to be down regulated by members of the miR-200 family (miR-200a,b,c miR-429, and miR-141).FindingsWe first used a computational target predicting system to identify 82 candidate miRNAs which bound the 3′UTR region of the Zeb-2 mRNA. Of these 82 miRNAs, precursors for 51 were available in our miRNA precursor library. Pre-miR™ Precursor Molecules for these 51 miRNAs were co-transfected into NIH3T3 cells with a luciferase reporter vector containing the 3′UTR region of the Zeb-2 mRNA. Seven miRNAs (miR-141, mi-183, miR-200a, miR-200b, miR-200c, miR-429 and miR-666-5p) were shown to down-regulate luciferase activity and Western blotting analysis confirmed that Pre-miR™ Precursor Molecules for these seven miRNAs induced expression of E-cadherin and miScript target protector against miR-183 and miR-666-5p abrogated this effect. Moreover, an Anti-miR™ miRNA Inhibitor targeting miR-183 and miR-666-5p repressed expression of E-cadherin.ConclusionsWe have established a method to identify miRNAs that bind the 3′UTR region of the Zeb-2 mRNA and that induce expression of E-cadherin, possibly by down-regulating the expression of Zeb-2. Our method may be more widely applicable for identifying miRNAs that bind target mRNA 3′UTR regions and down-regulate the expression of proteins encoded by these mRNAs.

Control of HCV Replication With iMIRs, a Novel Anti-RNAi Agent

MicroRNAs (miRNAs) serve important roles in regulating various physiological activities through RNA interference (RNAi). miR-122 is an important mediator of RNAi that is known to control hepatitis C virus (HCV) replication and is being investigated in clinical trials as a target for anti-HCV therapy. In this study, we developed novel oligonucleotides containing non-nucleotide residues, termed iMIRs, and tested their abilities to inhibit miR-122 function. We compared the inhibitory effects of iMIRs and locked nucleic acids (LNAs) on HCV replication in OR6 cells, which contained full-length HCV (genotype 1b) and a luciferase reporter gene. We found that RNA-type iMIRs with bulge-type, imperfect complementarity with respect to miR-122 were 10-fold more effective than LNAs in inhibiting HCV replication and functioned in a dose-dependent manner. Moreover, iMIR treatment of OR6 cells reduced HCV replication without inducing interferon responses or cellular toxicity. Based on these results, we suggest that iMIRs can inhibit HCV replication more effectively than LNAs and are therefore promising as novel antiviral agents.