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首页> 外文会议>International Symposium on Micro-NanoMechatronics and Human Science >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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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

机译：通过基于Epstein-Barr病毒的重组载体的转录因子和细胞因子基因体内的功能和功能丧失分析，以及它们对基因治疗和再生医学的新策略的意义

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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.
机译：体内遗传转移和调节方法对于分析和操纵细胞和组织功能至关重要。还应用了各种这种系统对基因治疗和再生医学等的基本和临床前研究，而临床利用率则到目前为止还没有成功。通过将eBV核抗原1（EBNA1）基因和orip衍生自EBV基因组（EBV基再生载体）并将它们与各种非病毒转染载体组合，通过插入EBV核抗原1（EBNA1）基因和ORIP来急剧改善非血管载体的基因转染效率。通过多尺度操纵程序，我们在将基于EBV的重组载体转染到哺乳动物细胞后，分析了EBNA1和ORIP序列的细胞内分布，并估计其对基于EBV的重组载体的高速率转染的意义。利用其在体内实现高效转基因的能力，我们还将基于EBV的再生载体系统应用于各种基因的体内功能分析，包括白细胞介素-21（IL-21）和IL-27。同时，RNA干扰（RNAi）提供了极其有用的工具，用于在各种生物中降低特定基因的表达。我们之前在体内哺乳动物器官中展示了RNAi，以及应用于各种疾病的治疗分子靶向，包括恶性黑素瘤和类风湿性关节炎。在这里，我们使用基于矢量EBV附加型转染和RNAi，以及它们在动物模型中的恶性疾病的控制有用的评估的体内功能性基因的分析本其它实例。 IL-23是IL-12家族细胞因子，在Th17分化中起着至关重要的作用。我们进行了IL-23编码EBV基再生载体的全身转染，并评估小鼠血清中细胞因子的浓度。当用预配的低免疫原性肿瘤转染压榨小鼠时，肿瘤的GO-WTH没有显着影响。同时，微蛋白相关的转录因子（MITF）基因在黑素瘤的存活和增殖中起不可或缺的作用。我们对在同性学小鼠移植的黑色素瘤中，在体内运动瘤中进行了电解介导的MITF表达的倒闭。结果，肿瘤的生长显着抑制，而使用EBV基EiPsomal载体将IL-12基因与肿瘤共传送到肿瘤中时，实现了更激烈的抑制。这些发现可能提出通过基于功能的功能和丧失功能丧失的基因体内功能分析的强大手段，并提出了新的基因治疗和免疫治疗策略来消除恶性肿瘤。

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