RNA-based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self-degradable lipid-like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self-degradability is based on a unique reaction "Hydrolysis accelerated by intra-Particle Enrichment of Reactant (HyPER)." In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid-scaffold lipid-like material that mounts all of these units (ssPalmO-Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.

Bone is one of the preferred sites for the metastasis of malignant tumours, such as breast cancer, lung cancer and malignant melanoma. Tumour cells colonizing bone have the capacity to induce the expression of receptor activator of nuclear factor-κB ligand (RANKL), which promotes osteoclast differentiation and activation. Tumour-induced osteoclastic bone resorption leads to a vicious cycle between tumours and bone cells that fuels osteolytic tumour growth, causing bone pain and hypercalcaemia. Furthermore, RANKL contributes to bone metastasis by acting as a chemoattractant to bone for tumour cells that express its receptor, RANK. Thus inhibition of the RANKL–RANK pathway is a promising treatment for bone metastasis, and a human monoclonal anti-RANKL antibody, denosumab, has been used in the clinic. However, orally available drugs targeting RANKL must be developed to increase the therapeutic benefits to patients. Here we report the efficacy of the small-molecule RANKL inhibitor AS2676293 in treating bone metastasis using mouse models. Oral administration of AS2676293 markedly inhibited bone metastasis of human breast cancer cells MDA-MB-231-5a-D-Luc2 as well as tumour-induced osteolysis. AS2676293 suppressed RANKL-mediated tumour migration in the transwell assay and inhibited bone metastasis of the murine cell line B16F10, which is known not to trigger osteoclast activation. Based on the results from this study, RANKL inhibition with a small-molecule compound constitutes a promising therapeutic strategy for treating bone metastasis by inhibiting both osteoclastic bone resorption and tumour migration to bone.

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Objective. To investigate T cell antigen receptor (TCR) clonotypes in rheumatoid arthritis (RA) lesions.Methods. Reverse transcriptase-polymerase chain reaction with TCR V, family-specific primers and subsequent single-strand conformation polymorphism (SSCP) analysis were performed. Direct nucleotide sequencing was also conducted.Results. A distinct clonal expansion of T cells was observed in the synovium. Furthermore, identical bands in samples of different areas of the same lesion were obtained by SSCP analysis. Nucleotide sequencing revealed that T cell clonotypes of identical mobility on SSCP analysis had the same nucleotide sequence and thus were identical clones. In 6 RA patients, &loo% of the expanded T cell clonotypes had identical migration patterns in 2 different samples, indicating that this percentage represents commonly existing T cell clonotypes in the affected joint. Furthermore, the Jg.1 gene segment was used predominantly by the TCR V, clonotypes that commonly expanded in the different portions of the same joint.Conclusion. These results suggest that the immune response in RA is not random, but rather is driven by common stimuli.

Gene therapy is a promising strategy for curing certain types of brain diseases. Supplementation of therapeutic proteins such as aromatic amino acid decarboxylase (AADC) or nerve growth factor (NGF) have been reported to be successful examples of such treatments. However, there are safety concerns because these systems are based on virus-based gene vectors. A safe and efficient artificial carrier is thus urgently needed as an alternative. In this study, an mRNA based artificial gene carrier was introduced into the mouse brain via intracerebroventricular administration. As a carrier, a lipid nanoparticle (LNP) composed of environmentally sensitive lipid-like materials called an SS-cleavable proton-activated lipid-like material is used. The apolipoprotein E mediated cellular uptake of the lipid nanoparticles is one of the key features for its superior and homogeneous transfection activity compared to commercially available transfection reagents in both in vitro and in vivo situations. Immunostaining of brain specimens suggested that exogenous proteins can be introduced into neuronal cells as well as astrocytes using the mRNA-based gene carrier. This cannot be achieved using DNA-based artificial gene carriers. The findings suggest that a combination of an mRNA and a lipid based delivery system have great promise as a platform for the treatment of brain disorders.

A renal cell carcinoma (RCC) is one of the refractory tumors, since it readily acquires resistance against chemotherapy. Thus, alternative therapeutic approaches such as obstructing the neovasculature are needed. We previously reported on the development of a plasmid DNA (pDNA)-encapsulating liposomal nanoparticle (LNP) as a hepatic gene delivery system that is applicable to systemic administration. The key molecular component is a SS-cleavable and pH-activated lipid-like material (ssPalm) that mounts dual sensing motifs (ternary amines and disulfide bonding) that are responsive to the intracellular environment. The main purpose of the present study was to expand its application to a tumor-targeting gene delivery systemin mice bearing tumors established from a RCC (OS-RC-2). When the modification of the surface of the particle is optimized for the polyethyleneglycol (PEG), stability in the blood circulation is improved, and consequently tumor-selective gene expression can be achieved. Furthermore, gene expression in the tumor was increased slightly when the hydrophobic scaffold of the ssPalm was replaced from the conventionally used myristic acid (ssPalmM) to alpha-tocopherol succinate (ssPalmE). Moreover, tumor growth was significantly suppressed when the completely CpG-free pDNA encoding the solute form of VEGFR (fms-like tyrosine kinase-1: sFlt-1) was used, especially when it was delivered by the LNP formed with ssPalmE(LNPssPalmE). Thus, the PEG-modified LNPssPalmE is a promising gene carrier for the cancer gene therapy of RCC. (C) 2014 Elsevier B.V. All rights reserved

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A lipid nanoparticle (LNP) composed of a series of SS-cleavable and pH-activated lipid-like materials (ssPalm) was previously developed as a platform of a gene delivery system. A tertiary amine and disulfide bonding were employed to destabilize the endosomal membrane and for intracellular collapse. We report herein on the development of a hepatocyte-targeting siRNA carrier by the molecular tuning of the hydrophobic scaffold, and tertiary amine structures. The gene knockdown activity against a hepatocyte-specific marker (factor VII: FVII) was improved when a more fat-soluble vitamin (vitamin E) was employed as a hydrophobic scaffold. Moreover, to allow the tertiary amines to accept protons by sensing a slight change in endosomal acidification, its structural flexibility was minimized by fixing it in a piperidine structure, and the distance between the surface of the particle to the ternary amine was increased. As a result, the pK a value was increased to the approximately 6.18 depending on its distance, while the pK a reached plateau when the tertiary amine was linked by an excess number of linear carbon chains. The pH-dependent membrane destabilization activity, as assessed by a hemolysis assay, was increased in parallel with the pK a value. Moreover, the gene knockdown activity was improved in parallel with hemolytic activity. Finally, further optimization of the lipid/siRNA ratio, and the use of chemically (2′-fluoro) modified siRNA synergistically improved the gene knockdown efficacy to an effective dose (ED 50 ) of 0.035 mg/kg. The developed ssPalm represents a promising platform for use as a hepatocyte-targeting siRNA carrier.