Nucleic Acid-Based Therapy: Development of a Nonviral-Based Delivery Approach

Gene therapy returns to the center stage of medicine to treat patients with diseases that are unable to be cured with the conventional therapeutic strategies. This development is due to various reasons, including vector development and significant achievement in next-generation sequencing. Among the various methodologies of gene therapy, nucleic acid-based therapy has been considered to be promising in various diseases. The development of delivery methods to target cells in vivo, however, remains critical. These include viral vector-based and nonviral vector-based gene delivery methods as well as physical approaches such as hydrodynamic gene delivery (HGD). HGD is a simple and effective in vivo gene transfer method for the functional analysis of therapeutic genes and regulatory elements in small animals. Moreover, this chapter outlines the principle of HGD, gene expression studies in rodents, and recent advances in clinical application of HGD and provides future perspectives in developing a safe and efficient method for nucleic acid-based therapy

NLRP3 Mediates NF-κB activation and cytokine induction in microbially induced and sterile inflammation

Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κ B. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interferencebased NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF- κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF- κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system

Low fat intake is associated with pathological manifestations and poor recovery in patients with hepatocellular carcinoma

BACKGROUND: This study aimed to clarify whether dietary deviation is associated with pathological manifestations in hepatocellular carcinoma (HCC) patients. METHODS: Dietary intake was estimated in 35 HCC cases before and after hospitalization by referencing digital camera images of each meal. Pathological conditions were evaluated in nitrogen balance, non-protein respiratory quotient (npRQ), neuropsychiatric testing and recovery speed from HCC treatment. RESULTS: On admission, nitrogen balance and npRQ were negative and less than 0.85, respectively. Five patients were judged to have suffered from minimal hepatic encephalopathy that tended to be associated with a lowered value of npRQ (p = 0.082). The energy from fat intake showed a tendency of positive correlation with npRQ (p = 0.11), and the patients with minimal hepatic encephalopathy took significantly fewer energy from fat (p = 0.024). The energy difference from fat between diets at home versus those in the hospital showed a significant positive correlation with npRQ change after admission (p = 0.014). The recovery speed from invasive treatments for HCC showed a significant negative correlation with npRQ alteration after admission (p = 0.0002, r = −0.73). CONCLUSIONS: These results suggest the lower fat intake leads to deterioration of energy state in HCC patients, which associates with poor recovery from invasive treatments and various pathological manifestations

Oral delivery of Lactococcus lactis that secretes bioactive heme oxygenase-1 alleviates development of acute colitis in mice

Background: Mucosal delivery of therapeutic proteins using genetically modified strains of lactic acid bacteria (gmLAB) is being investigated as a new therapeutic strategy. Methods: We developed a strain of gmLAB, Lactococcus lactis NZ9000 (NZ-HO), which secretes the anti-inflammatory molecule recombinant mouse heme oxygenase-1 (rmHO-1). The effects of short-term continuous oral dosing with NZ-HO were evaluated in mice with dextran sulfate sodium (DSS)-induced acute colitis as a model of inflammatory bowel diseases (IBD). Results: We identified the secretion of rmHO-1 by NZ-HO. rmHO-1 was biologically active as determined with spectroscopy. Viable NZ-HO was directly delivered to the colon via oral administration, and rmHO-1 was secreted onto the colonic mucosa in mice. Acute colitis in mice was induced by free drinking of 3 % DSS in water and was accompanied by an increase in the disease activity index score and histopathological changes. Daily oral administration of NZ-HO significantly improved these colitis-associated symptoms. In addition, NZ-HO significantly increased production of the anti-inflammatory cytokine interleukin (IL)-10 and decreased the expression of pro-inflammatory cytokines such as IL-1 alpha and IL-6 in the colon compared to a vector control strain. Conclusions: Oral administration of NZ-HO alleviates DSS-induced acute colitis in mice. Our results suggest that NZ-HO may be a useful mucosal therapeutic agent for treating IBD.ArticleMICROBIAL CELL FACTORIES. 14:189 (2015)journal articl

Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity

The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl-YVAD- chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Activation of ASC induces apoptosis or necrosis, depending on the cell type, and causes tumor eradication

The adaptor protein ASC (also called TMS1) links certain NLR proteins (e.g., NLRC4, NLRP3) and caspases. It is involved in the chemosensitivity of tumor cells and inflammation. Here, we found that ASC activation using NLRC4 mimicry or an autoinflammatory disease-associated NLRP3 mutant induced necrosis in COLO205 colon adenocarcinoma cells, but induced caspase-8-dependent apoptosis in NUGC-4 stomach cancer cells. As the Fas ligand induced caspase-8-dependent apoptosis in COLO205 cells, caspase-8 was intact in this cell line. ASC-mediated necrosis was preceded by lysosomal leakage, and diminished by inhibitors for vacuolar H+-ATPase, cathepsins, and calpains but not by inhibitors for caspase-8, or aspartic proteases, suggesting that lysosomes and certain proteases were involved in this process. Finally, growing tumors of transplanted human cancer cells in nude mice were eradicated by the activation of endogenous ASC in the tumor cells, irrespective of the form of cell death. Thus, ASC mediates distinct forms of cell death in different cell types, and is a promising target for cancer therapy. (Cancer Sci 2010). © 2010 Japanese Cancer Association