Hidefumi Mukai

Renal press-mediated transfection method for plasmid DNA and siRNA to the kidney

Gene and oligonucleotides transfection methods to the kidney are required for the progress of biomedical research and the therapy of renal diseases. In this study, we found that siRNA as well as plasmid DNA can be transfected to the kidney by a simple method including lightly and once pressing the kidney after intravenous injection of siRNA or plasmid DNA (renal press-mediated transfection method). Using luciferase as the reporter, gene expression and silencing properties were evaluated. Plasmid DNA is efficiently and widely transfected to the periphery of the pressed kidney, and also siRNA is transfected into the kidney and significant suppression of gene expression can be achieved. Additively, serum creatinine and blood urea nitrogen levels, that are indices of renal function, exhibited no marked changes after transfection by this method. Therefore, it appears that plasmid DNA and siRNA could be transfected to the kidney without renal dysfunction by renal press-mediated transfection method.

Pressure-Mediated Transfection of Murine Spleen and Liver

Extension of in vivo nucleic acid transfection techniques and increased information about those transfection properties and side effects are urgently needed to advance biological research and drug therapy. Tissue pressure-mediated transfection, involving lightly pressing the target tissue after intravenous injection of plasmid DNA or small-interfering RNA (siRNA), is a promising approach because of its high transfection efficiency and resulting low tissue damage. In this study, the gene expression/silencing properties and proinflammatory cytokine production associated with tissue pressure-mediated transfection were evaluated to extend its application. We have found that tissue pressure-mediated transfection can be applied to plasmid DNA and siRNA transfection to the spleen and siRNA transfection to the liver. In addition, we have demonstrated that these methods induce little production of proinflammatory cytokines, including tumor necrosis factor-alpha, interleukin (IL)-6, IL-12, and interferon-gamma. Moreover, we succeeded in controlling and quantifying the degree of pressure on the spleen and kidney and found that 0.59 N/cm(2) is sufficient for efficient and highly reproducible plasmid DNA transfection to the spleen and kidney in mice. Tissue pressure-mediated transfection of the kidney, liver, and spleen exhibits well-balanced characteristics including (1) simple and convenient manipulation, (2) tissue-specific, effective broad transfection properties, and (3) a low inflammatory response. Therefore, this information could be useful for a molecular-level mechanism analysis of diseases at an individual level in mammals, exploration of therapeutic target molecules and evaluation of gene therapy and nucleic acid-based therapy approaches, as well as potential clinical applications.

Long-term in vivo gene expression in mouse kidney using φC31 integrase and electroporation

Abstract Background: Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes. Purpose: The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using ϕC31 integrase. Methods: Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5 µg of pORF-luc/attB as a donor plasmid and 20 µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression. Results: Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, ϕC31 integrase-transfected cells had less genomic DNA damage caused by integrase expression. Discussion and conclusion: These results demonstrated that the ϕC31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.

Urokinase injection-triggered clearance enhancement of a 4-arm PEG-conjugated 64 Cu-bombesin analog tetramer: A novel approach for the improvement of PET imaging contrast

ABSTRACT Radiolabeled antibodies, polyethylene glycol‐conjugated (PEGylated) peptides, liposomes, and other materials were investigated as positron‐emission tomography (PET) probes. These substances accumulate in tumors but often remain too long in circulation. We investigated the combination of intravenous urokinase injection and its substrate linker as a triggered radioisotope clearance enhancement system to improve imaging contrast. To this end, we synthesized a four‐arm PEGylated 64Cu‐bombesin analog tetramer with a urokinase substrate linker. In mouse blood, it was almost perfectly cleaved and degraded into smaller radioactive fragments in vitro with urokinase (≥20,000IU/mL). In mouse blood circulation, ˜50–65% of the probe was rapidly degraded after the urokinase injection and the radioactive fragments were eliminated mainly from the kidney. In contrast, tumor radioactivity levels did not change, and therefore, the tumors were clearly visualized. The tumor/blood ratio, an indicator of imaging contrast, increased 2.5 times, while elimination of the radioisotope from the blood was enhanced. This approach has the potential to improve imaging contrast using various PET probes. It could also shorten the time required to obtain sufficient contrast and decrease patient radiation exposure.

Expanding the Applicability of the Metal Labeling of Biomolecules by the RIKEN Click Reaction: A Case Study with Gallium-68 Positron Emission Tomography

Radiolabeled biomolecules with short half‐life times are of increasing importance for positron emission tomography (PET) imaging studies. Herein, we demonstrate an improved and generalized method for synthesizing a [radiometal]‐unsaturated aldehyde as a lysine‐labeling probe that can be easily conjugated into various biomolecules through the RIKEN click reaction. As a case study, 68Ga‐PET imaging of U87MG xenografted mice is demonstrated by using the 68Ga‐DOTA‐RGDyK peptide, which is selective to αVβ3 integrins.

Potential usefulness of Brevibacillus for bacterial cancer therapy: intratumoral provision of tumor necrosis factor-α and anticancer effects

Bacterial cancer therapy, wherein bacteria are used as a gene expression system for the exogenous protein of interest in the body, has started becoming a focus area of research; therefore, studying potential bacterial species for use is extremely important. Here, we investigated the use of Brevibacillus choshinensis as an effective and safe provider of anticancer proteins in the body, using a transformant expressing murine tumor necrosis factor-α (mTNF-α). The transformant sustainably provided mTNF-α in tumors in mice for a few hours post-injection. The growth of TNF-α-sensitive tumors was inhibited even by the control transformant, which did not provide mTNF-α; intratumoral mTNF-α provision by Brevibacillus choshinensis had additive effects on tumor growth inhibition. In contrast, intratumorally injected recombinant mTNF-α did not inhibit tumor growth because of rapid elimination from the tumor. Blood biochemical and histochemical analyses showed that intravenous injection of the transformant that did not provide mTNF-α did not lead to tissue injury and dysfunction or infiltration of inflammatory cells over 1 week. Considering the findings, this approach is expected to have a high degree of usability as a delivery system for protein pharmaceuticals, especially from the viewpoints of loading capacity and cost effectiveness.