Gene therapy for gastric diseases.

Gene therapy for gastric cancer and gastric ulcer is a rationalized strategy since various genes correlate with these diseases. Since gene expressions in non-target tissues/cells cause side effects, a selective gene delivery system targeted to the stomach and/or cancer must be developed. The route of vector transfer (direct injection, systemic, intraperitoneal, gastric serosal surface and oral administration) is an important issue which can determine efficacy and safety. Strategies for cancer gene therapy can be categorized as suicide gene therapy, growth inhibition and apoptosis induction, immunotherapy, anti-angiogenesis, and others. Combination of the target gene with other genes and/or strategies such as chemotherapy and virotherapy is promising. Candidates for treatment of gastric ulcer are vascular endothelial growth factor, angiopoietin-1, serum response factor, and cationic host defense peptide cathelicidin. In this review, we discuss stomach- and cancer-targeted gene transfer methods and summarize gene therapy trials for gastric cancer and gastric ulcer

Flavonoids Enhance Lipofection Efficiency and Ameliorate Cytotoxicity in Colon26 and HepG2 Cells via Oxidative Stress Regulation

The generation of reactive oxygen species (ROS) can affect cationic liposome-mediated transfection. In this study, we focused on a specific class of antioxidants, flavonoids, to investigate the transfection efficiency using cationic liposome/plasmid DNA complexes (lipoplexes) in 2D and 3D cultures of Colon26 and HepG2 cells, respectively. All tested flavonoids enhanced the transfection efficiency in 2D Colon26 and HepG2 cells. Among the tested flavonoids, 25 µM quercetin showed the highest promotion effect of 8.4- and 7.6-folds in 2D Colon26 and HepG2 cells, respectively. Transfection was also performed in 3D cultures of Colon26 and HepG2 cells using lipoplexes with quercetin. Quercetin (12.5 µM) showed the highest transfection efficiency at all transfection timings in 3D Colon26 and HepG2 cells with increased cell viability. Flow cytometry revealed that quercetin treatment reduced the population of gene expression-negative cells with high ROS levels and increased the number of gene expression-positive cells with low ROS levels in HepG2 cells. Information from this study can be valuable to develop strategies to promote transfection efficiency and attenuate cytotoxicity using lipoplexes

Suppression of Peritoneal Fibrosis by Sonoporation of Hepatocyte Growth Factor Gene-Encoding Plasmid DNA in Mice

Gene therapy is expected to be used for the treatment of peritoneal fibrosis, which is a serious problem associated with long-term peritoneal dialysis. Hepatocyte growth factor (HGF) is a well-known anti-fibrotic gene. We developed an ultrasound and nanobubble-mediated (sonoporation) gene transfection system, which selectively targets peritoneal tissues. Thus, we attempted to treat peritoneal fibrosis by sonoporation-based human HGF (hHGF) gene transfection in mice. To prepare a model of peritoneal fibrosis, mice were intraperitoneally injected with chlorhexidine digluconate. We evaluated the preventive and curative effects of sonoporation-based hHGF transfection by analyzing the following factors: hydroxyproline level, peritoneum thickness, and the peritoneal equilibration test. The transgene expression characteristics of sonoporation were also evaluated using multicolor deep imaging. In early-stage fibrosis in mice, transgene expression by sonoporation was observed in the submesothelial layer. Sonoporation-based hHGF transfection showed not only a preventive effect but also a curative effect for early-stage peritoneal fibrosis. Sonoporation-based hHGF transfection may be suitable for the treatment of peritoneal fibrosis regarding the transfection characteristics of transgene expression in the peritoneum under fibrosis

Absorption characteristics of model compounds from the small intestinal serosal surface and a comparison with other organ surfaces

We examined the absorption of phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextrans (FD-4, MW 4,400; FD-10, MW 9,500; FD-40, MW 40,500) as model compounds through the small intestinal serosal surface. After application to the rat small intestinal serosal surface using a cylindrical diffusion cell, each compound was absorbed at different rates. The absorption ratios in 6 h after PSP, FD-4, FD-10 and FD-40 application were calculated to be 89.2, 34.6, 14.9 and 2.1 % of dose, respectively. Elimination profiles of PSP, FD-4 and FD-10 from the small intestinal serosal surface obeyed first-order kinetics. Moreover, we calculated the apparent permeability coefficient Papp for comparison to other organ surfaces. The kidney had the highest absorption efficiency, as shown by having more than 1.5 times significantly higher Papp values of PSP, FD-4 and FD-10. Similar to the other organ surfaces, a correlation was observed between the Papp of small intestine and the molecular weight of these hydrophilic compounds. In addition, the small intestine is likely to contribute largely to hydrophilic compounds absorption from the peritoneal cavity, judging from absorption clearance CLa calculated by utilizing the peritoneal organ surface area

Understanding In Vivo Fate of Nucleic Acid and Gene Medicines for the Rational Design of Drugs

Nucleic acid and genetic medicines are increasingly being developed, owing to their potential to treat a variety of intractable diseases. A comprehensive understanding of the in vivo fate of these agents is vital for the rational design, discovery, and fast and straightforward development of the drugs. In case of intravascular administration of nucleic acids and genetic medicines, interaction with blood components, especially plasma proteins, is unavoidable. However, on the flip side, such interaction can be utilized wisely to manipulate the pharmacokinetics of the agents. In other words, plasma protein binding can help in suppressing the elimination of nucleic acids from the blood stream and deliver naked oligonucleotides and gene carriers into target cells. To control the distribution of these agents in the body, the ligand conjugation method is widely applied. It is also important to understand intracellular localization. In this context, endocytosis pathway, endosomal escape, and nuclear transport should be considered and discussed. Encapsulated nucleic acids and genes must be dissociated from the carriers to exert their activity. In this review, we summarize the in vivo fate of nucleic acid and gene medicines and provide guidelines for the rational design of drugs

Highly stomach-selective gene transfer following gastric serosal surface instillation of naked plasmid DNA in rats.

BACKGROUND: The purpose of this study was to achieve stomach-selective gene transfer in rats by our simple and novel administration method, which is gastric serosal surface instillation of naked plasmid DNA (pDNA). METHODS: Naked pDNA encoding firefly luciferase as a reporter gene was instilled onto the gastric serosal surface in male Wistar rats. As controls, we performed intraperitoneal, intragastric and intravenous administration of naked pDNA. At appropriate time intervals, we measured luciferase activities in the stomach and other tissues. RESULTS: Gene expression in the stomach 6 h after gastric serosal surface instillation of naked pDNA (5 microg) was significantly higher than that after using other administration methods. The present study is the first report on stomach-selective gene transfer following instillation of naked pDNA onto the gastric serosal surface in rats. Also, the gene expression level in the stomach 6 h after gastric serosal surface instillation of naked pDNA was markedly higher than that in other tissues. In a dose-dependent study, the gene expression level was saturated over 5 microg. Gene expression in the stomach was detected 3 h after gastric serosal surface instillation of naked pDNA. The gene expression level peaked 12-24 h after instillation of naked pDNA, then decreased to a level similar to 3 h at 48 h. CONCLUSIONS: Gastric serosal surface in stillation of naked pDNA can be a highly stomach-selective gene transfer method in rats

Effective intraperitoneal gene transfection system using nanobubbles and ultrasound irradiation

In this study, we demonstrate the low toxicity and highly efficient and spatially improvedtransfection of plasmid DNA (pDNA) with liposomal nanobubbles (bubble liposomes [BLs])using ultrasound (US) irradiation in mice. Naked pDNA with BLs was intraperitoneally injected, followed by US irradiation. The injection volume, the duration of US irradiation, and the dose of BLs were optimized. Both BLs and US irradiation were essential to achieve high transgeneexpression from naked pDNA. We observed transgene expression in the entire peritonealtissues, including the peritoneal wall, liver, spleen, stomach and small and large intestines. The area of transfection could be controlled with focused US irradiation. There were few changes in the morphology of the peritoneum, the peritoneal function or serum alanine aminotransferase levels, suggesting the safety of BLs with US irradiation. Using a tissue-clearing method, the spatial distribution of transgene expression was evaluated. BLs with US irradiation delivered pDNA to the submesothelial layer in the peritoneal wall, whereas transgene expression was restricted to the surface layer in the liver and stomach. Therefore, BLs with USirradiation could be an effective and safe method of gene transfection to the peritoneum

Monitoring method for transgene expression in target tissue by blood sampling

In this study, we have developed a novel method to monitor transgene expression in tissues by blood sampling. We administered plasmid DNA (pDNA) encoding non-secretory form of firefly luciferase as a reporter gene and pDNA encoding secretable Gaussia princeps luciferase as a monitor gene simultaneously into mice. Good positive correlations were found between log-transgene expression of the reporter gene and the monitor gene in the treated muscle, between the monitor gene in the treated muscle and plasma, and consequently between the reporter gene in the treated muscle and the monitor gene in plasma after naked pDNA transfer into the muscle of mice. Such positive correlations were also found with gastric serosal surface instillation of naked pDNA, intravenous injection of lipoplex, and hydrodynamics-based injection of naked pDNA. We developed monitoring method of transgene expression in tissues by blood sampling, which was named ‘Therapeutic transgene monitoring (TTM)’, after ‘Therapeutic drug monitoring (TDM)’

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

We have previously developed an efficient and safe transfection method for the kidney in mice: renal suction-mediated transfection. In this study, we verified the detailed characteristics of transgene expression and plasmid DNA (pDNA) in mice to develop therapeutic strategies and application to gene function analysis in the kidney. After naked pDNA was administered intravenously, the right kidney was immediately suctioned by a tissue suction device. We examined the spatial distribution of transgene expression and pDNA in the suctioned kidney using tissue clearing by CUBIC, ClearT2, and Scale SQ reagents. Spatial distribution analysis showed that pDNA was transfected into extravascular cells and sufficiently delivered to the deep renal cortex. In addition, we revealed that transgene expression occurred mainly in peritubular fibroblasts of the suctioned kidney by tissue clearing and immunohistochemistry. Next, we confirmed the periods of pDNA uptake and activation of transcription factors nuclear factor-κB and activator protein 1 by luciferase assays. Moreover, the use of a pCpG-free plasmid enabled sustained transgene expression in the suctioned kidney. In conclusion, analyses of the spatial distribution and immunostaining of the section suggest that pDNA and transgene expression occurs mainly in peritubular fibroblasts of the suctioned kidney. In addition, we clarified some factors for efficient and/or sustained transgene expression in the suctioned kidney