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Gain-of-function and loss-of-function analyses in vivo of transcriptional factor and cytokine genes using Epstein-Barr virus-based episomal vectors, and their implication to novel strategies of gene therapy and regenerative medicine

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11 Author(s)
Kishida, T. ; Depts. of Microbiol. & Immunology, Kyoto Prefectural Univ. of Med., Kyoto, Japan ; Nakai, N. ; Matsui, M. ; Yoshimoto, K.
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Genetic transfer and regulation methodologies in vivo are essential for analysis and manipulation of cells and tissue functions. A variety of such systems have also been applied to basic and preclinical studies on gene therapy and regenerative medicine, etc., while clinical utilization has not been successful enough so far. The gene transfection efficiencies of nonviral vectors are drastically improved by inserting EBV nuclear antigen 1 (EBNA1) gene and oriP that are derived from EBV genome into the expression vectors (EBV-based episomal vectors) and combining them with various non-viral transfection vehicles. By means of the multiscale manipulation procedures, we analyzed intracellular distribution of EBNA1 and oriP sequence after transfected an EBV-based episomal vector into mammalian cells, and estimated its implication to the high rate transfection of the EBV-based episomal vectors. Taking advantage of its ability to achieve highly efficient transfecton in vivo, we also applied the EBV-based episomal vector system for in vivo functional analyses of variety of genes, including interleukin-21 (IL-21) and IL-27. Meanwhile, RNA interference (RNAi) offers extremely useful tools for down-regulating expression of specific genes in a variety of organisms. We previously showed RNAi in mammalian organs in vivo, as well as its application to therapeutic molecular targeting of various disorders including malignant melanoma and rheumatoid arthritis. Here we present other examples of functional gene analyses in vivo using the EBV-episomal vector-based transfection and RNAi, and of assessment of their usefulness in control of malignant diseases in animal models. IL-23 is an IL-12 family cytokine and plays a crucial role in Th17 differentiation. We performed systemic transfection of IL-23-encoding EBV-based episomal vector and evaluated concentration of the cytokine in mouse serum. When the plasmid was transfected into the mice with preestablished low-immunogenic tumor, the gro- - wth of the tumor was not significantly influenced. Meanwhile, microphthalmia-associated transcription factor (Mitf) gene plays indispensable roles in survival and proliferation of melanoma. We performed electro-poration-mediated knock-down of Mitf expression in vivo in melanoma that was transplanted in syngenic mice. As results, the growth of the tumor was significantly suppressed, while more drastic suppression was achieved when IL-12 gene was co-electrotransfected into the tumor using an EBV-based eipsomal vector. These findings may suggest powerful means for in vivo functional analyses of genes through both gain-of-function- and loss-of-function-based procedures, and propose novel gene therapy and immunotherapy strategies to eradicate malignancies.

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Micro-NanoMechatronics and Human Science (MHS), 2010 International Symposium on

Date of Conference:

7-10 Nov. 2010

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