Tadaaki Ohgi

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.

Signalling mechanisms in sphingosine 1‐phosphate‐promoted mesangial cell proliferation

Background: The bioactive sphingolipid sphingosine 1‐phosphate (S1P) is formed by the activation of sphingosine kinase (SPHK) in diverse stimuli, such as platelet‐derived growth factor (PDGF). S1P acts not only as an extracellular mediator but also as an intracellular second messenger, resulting in the proliferation of various different types of cells. However, the signal transduction mechanism in S1P‐induced proliferation of mesangial cells is poorly known.

Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications.

Global analysis of differentially expressed genes during progression of calcium oxalate nephrolithiasis

The process of nephrolithiasis development is poorly understood at the molecular level. Here, we constructed a cDNA microarray from a rat kidney normalized cDNA library, and investigated the pattern of gene expression in rat kidneys from a calcium oxalate (CaOx) nephrolithiasis model. One hundred and seventy-three genes were found to be at least 2-fold regulated at one or more time points during progression of nephrolithiasis. RT-PCR and immunohistochemical analyses confirmed differential expression at both transcriptional and translational levels of genes identified by cDNA microarray screening. The differentially regulated genes were grouped into six clusters based on their expression profiles; the magnitude and the temporal patterns of gene expression identified known and novel molecular components involved in inflammation and matrix expansion in the CaOx nephrolithiasis kidney. This microarray study is the first report on gene expression programs underlying the process of nephrolithiasis.

Crystal structure of human ISG20, an interferon-induced antiviral ribonuclease.

ISG20 is an interferon‐induced antiviral exoribonuclease that acts on single‐stranded RNA and also has minor activity towards single‐stranded DNA. It belongs to the DEDDh group of RNases of the DEDD exonuclease superfamily. We have solved the crystal structure of human ISG20 complexed with two Mn2+ ions and uridine 5′‐monophosphate (UMP) at 1.9 Å resolution. Its structure, including that of the active site, is very similar to those of the corresponding domains of two DEDDh‐group DNases, the ε subunit of Escherichia coli DNA polymerase III and E. coli exonuclease I, strongly suggesting that its catalytic mechanism is identical to that of the two DNases. However, ISG20 also has distinctive residues, Met14 and Arg53, to accommodate hydrogen bonds with the 2′‐OH group of the UMP ribose, and these residues may be responsible for the preference of ISG20 for RNA substrates.

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.

Functional genomic search of G-protein-coupled receptors using microarrays with normalized cDNA library

G-protein-coupled receptors (GPCRs) represent the single most important drug targets for medical therapy, and information from genome sequencing and genomic databases has substantially accelerated their discovery. Despite its present large size, the GPCR superfamily continues to expand rapidly as new receptors are discovered through automated sequencing of cDNA libraries and bioinformatics. It is estimated that several thousand GPCRs may exist in the human genome, and, at present, with most of the genome sequenced, as many as 250 GPCRs have been cloned. However, a systematic approach to identify the function of newly discovered GPCRs is lacking. Large-scale monitoring of gene expression is a powerful approach for clarifying cellular events. DNA microarray technologies permit the recognition of genome-wide expression profiling, and have a profound impact to biological research, especially pharmacology. This technology can also be applied to drug discovery and molecular classification of diseases.

Transcriptional profiling of gene expression patterns during sphingosine 1-phosphate-induced mesangial cell proliferation.

Sphingosine 1-phosphate (S1P) is known to regulate cell proliferation, apoptosis, and motility. Recently, we have reported that S1P and its analogue dihydro-S1P (DHS1P) promote proliferation of rat cultured mesangial cells. To investigate the signaling mechanisms underlying S1P- and DHS1P-induced mesangial cell proliferation, we performed cDNA microarray analysis of gene expression during mesangial cell proliferation. In terms of the overall pattern, gene expression waves induced by S1P and DHS1P were similar to those induced by a potent mesangial mitogen platelet-derived growth factor (PDGF), whereas we found several genes, such as two growth factors, connective tissue growth factor (CTGF) and heparin-binding EGF-like growth factor (HB-EGF), which were induced by the sphingolipids, but not by PDGF. Cluster analysis also identified calcium-dependent molecules highly expressed in DHS1P-stimulated cells compared to S1P-stimulated cells. Calcium mobilization analysis showed that DHS1P had higher magnitudes of intracellular calcium mobilization than S1P, suggesting that S1P and DHS1P differentially regulate intracellular calcium mobilization, possibly leading to different gene expression in mesangial cells. The large-scale monitoring of gene expression performed here allows us to identify S1P-induced transcriptional properties during mesangial cell proliferation.

Role of Chk1 and Chk2 in Ara-C-induced differentiation of human leukemia K562 cells.

Human chronic myelogenous leukemia K562 cells are relatively resistant to the anti‐metabolite cytosine arabinoside (Ara‐C) and, when treated with Ara‐C, they differentiate into erythrocytes without undergoing apoptosis. In this study we investigated the mechanism by which Ara‐C induces K562 cells to differentiate. We first observed that Ara‐C‐induced differentiation of these cells is completely inhibited by the radiosensitizing agent caffeine, an inhibitor of ATM and ATR protein kinases. We next found that Ara‐C activates Chk1 and Chk2 in the cells, and that the activation of Chk1, but not of Chk2, was almost completely inhibited by caffeine. Proteasome‐mediated degradation of Cdc25A and phosphorylation of Cdc25C were induced by Ara‐C treatment, presumably due to the activation of Chk2 and Chk1, respectively. To directly observe the effects of checkpoint kinase activation in Ara‐C‐induced differentiation, we suppressed Chk1 or Chk2 with the Chk1‐specific inhibitor Gö6976, by generating cell lines stably over‐expressing dominant‐negative forms of Chk2, or by siRNA‐mediated knock‐down of the Chk1 or the Chk2 gene. The results suggest that Ara‐C‐induced erythroid differentiation of K562 cells depends on both Chk1 and Chk2 pathways.

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.