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

Non-viral vectors are considered to be an attractive approach for gene delivery, since an artificial material is less immunogenic and oncogenic compared to a viral vector. We previously reported on the hepatic delivery of plasmid DNA (pDNA) by using lipid-like material (an SS-cleavable and pH-activated lipid-like material: ssPalm) which mounts two hydrophobic scaffolds, proton-accepting motifs (tertiary amines), and a cleavable unit (disulfide bonding). In the present study, we report on an advanced hepatic gene delivery system that uses a new type of ssPalm derivative: ssPalmE-Paz4-C2. The hepatic transgene expression of the intravenously administrated lipid nanoparticle (LNP) that was formed with the ssPalmE-Paz4-C2 (LNP) was significantly higher than that of conventional LNPs formed with a myristic acid-scaffold ssPalm (LNP). However, the LNP particle induced a severe innate immune response that involved the production of the pro-inflammatory cytokines (IL-6 and TNFα), intracellular DNA sensor-related cytokine (IL-1β) and interferon (IFNβ), even when a pDNA free from CpG-motifs was encapsulated. The production of the pro-inflammatory cytokines and the DNA sensor-related cytokines is attributed to the combination of vitamin E scaffolds and encapsulated pDNA. The depletion of macrophages by chlodronate-encapsulating liposomes dramatically reduced inflammatory gene expression. Based on the above findings, we conclude that the use of a certain type of non-viral carrier that shows a robust gene expression activity is attended by a risk of eliciting an innate immune response. When a highly hydrophobic derivative of dexamethasone, an anti-inflammatory glucocorticoid compound, was co-loaded to the particle, this inflammatory response was relieved, and gene expression efficiency was enhanced. It is thus concluded that the co-delivery of dexamethasone and pDNA is a promising approach for reducing these risks.

A positive feedback system, using GAL4-vp16 (a fusion protein of yeast GAL4 and herpes simplex virus vp16) as an activator and firefly luciferase as a reporter, maintained luciferase expression for 7 d in mice. However, the luciferase expression decreased after 7 d, and this phenomenon could be caused by immunoreactions against these exogenous proteins. This hypothesis was examined by the following three strategies, designed to avoid the putative immunoreactions: (i) use of the endogenous secreted alkaline phosphatase (SEAP) protein as a reporter, (ii) replacement of vp16 with endogenous transcription factors, and (iii) insertion of the target sequence of microRNA expressed in cells of hematopoietic origin, to suppress GAL4-vp16 expression in antigen-presenting cells. The results obtained in this study suggested that silencing would be induced by mechanism(s) besides immunoreactions against reporter and activator proteins.

The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA.

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