Bridge Of Nucleic Acids Chemistry (Japan)

Rationale] The pathogenesis of pulmonary fibrosis is complex because multiple pathways and molecules are speculated to be associated with the disease. Given this complexity of the pathogenic processes, the combination therapy may be a better approach than the current standard therapy. Despite transforming growth factor-1 (TGF-1) is a powerful activator of connective tissue synthesis and fibroblast proliferation in the lung, nintedanib, one of the two approved drugs for the treatment of idiopathic pulmonary fibrosis, does not directly inhibit the TGF-b1 signaling pathway. TRK-250 targeting human TGF-1 has received much attention as a novel treatment for idiopathic pulmonary fibrosis. PK-7010 is a novel oligonucleotide therapeutics that selectively and directly inhibits the expression of mouse TGF-b1 at RNA level. Therefore, in this study, we evaluated the synergistic effect of combined therapy using PK-7010 and nintedanib on experimental pulmonary fibrosis in mice.

Highly stable PEGylated cationic hybrid bicellar nanodisc for efficient siRNA delivery.

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of 18O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging 18O atom into the phosphate group during the oxidation step of the synthetic cycle by using 18O water as the oxygen donor. The 18O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The 18O/16O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of 18O-labeled RNA, and this technique was used to determine the blood concentration of 18O-labeled RNA after administration to mice. 18O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.

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.

Current criteria for a reactive (positive) interpretation on hepatitis C virus (HCV) recombinant immunoblot assay (RIBA) require > or = 1+ reactivity to at least two of the four HCV antigens present in the assay. Given that 5-1-1 is a subcomponent of c100-3, there is concern that donor samples reacting only with these two antigens (and not with c22-3 or c33c) could be incorrectly classified as positive on the basis of limited reactivity to only one HCV gene product. It is determined that 0.23 to 0.44 percent of HCV enzyme immunoassay-repeatably reactive donor sera demonstrate a pattern of 5-1-1 and c100-3 only on RIBA. Evaluation of six such donor sera using peptide enzyme immunoassays spanning the c100-3 antigen showed highly restricted reactivity to the 5-1-1 N-terminal region of c100-3, in contrast to broad 5-1-1 and c100-3 C-terminal peptide reactivity observed in the majority of donor sera with other positive RIBA patterns. HCV polymerase chain reaction and follow-up serologic evaluations of four of these donors indicated the absence of viremia or evolving seroconversion in all cases. It is concluded that, in the blood donor setting, a pattern of only 5-1-1 and c100-3 reactivity is typically not indicative of HCV infection. To avoid overinterpretation, it is recommended that RIBA grading criteria be revised to require reactivity to two or more HCV-encoded gene products.