Cytokine gene therapy of cardiovascular diseases by in vivo electroporation

体内电穿孔细胞因子基因治疗心血管疾病

• 批准号: 13557063
• 负责人: MATSUMORI Akira
• 金额: $ 8.51万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557063/
• 关键词: gene; electroporation; cytokine; mouse; myocarditis; stem cell factor; c-kit; gene therapy; 心疾患; ウイルス; IL-1; IL-10

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe.We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral KL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle, Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL-1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak.Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL-1ra or vIL-10 express … More ion plasmid had significantly improved the survival rates of the animals. The expression of TNF-α was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ran treatment, and the expression of IFN-γ in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls.To increase the efficacy of gene transfer, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated vIL-10 expression in vivo. The fusion cytokine vIL-10/mutFC was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine decreased the mortality in a mouse viral myocarditis model. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein gene. Less

在众多的体内基因转移技术中，直接肌肉注射质粒DNA是一种简单、廉价和安全的方法。我们尝试通过体内电穿孔法将抑制性细胞因子IL-1受体拮抗剂（IL-1 ra）或病毒KL-10（vIL-10）导入肌肉，用10 PFU的脑心肌炎病毒腹膜内接种4周龄雄性DBA/2小鼠。病毒接种后，立即将携带IL-1 ra或vIL-10的表达质粒注射到胫骨前肌中，随后进行电穿孔。IL-1 ra和vIL-10在第5天达到高峰，血清IL-1 ra和vIL-10水平分别为10.5和2.3ng/ml，心肌细胞浸润较对照组明显减轻。在心肌炎发病后第14天，IL-1 ra或vIL-10表达的转移 ...更多信息 离子质粒能显著提高动物的存活率。TNF-α的表达下降至0.60倍（p < 0.005）和诱导型一氧化氮合酶（iNOS）0.43倍（p < 0.005），心脏IFN-γ的表达降低至对照组的0.35倍（p < 0.05），和iNOS 0.21倍（p < 0.005）。为了增加基因转移的功效，我们开发了一种质粒载体，其表达分泌蛋白作为与非溶细胞免疫球蛋白Fc部分的融合蛋白，并将其用于电穿孔-介导的vIL-10表达。成功表达了融合细胞因子vIL-10/mutFC，并且vIL-10的峰值血清浓度几乎是非融合vIL-10表达质粒的100倍（195 ng/ml）。表达的融合细胞因子降低小鼠病毒性心肌炎模型的死亡率。这些结果表明，表达非细胞溶解性Fc融合细胞因子的质粒DNA的转移可用于递送增强水平的细胞因子而不改变一般生物活性。这一简单、高效的系统为人类疾病的基因治疗提供了一条新的途径，并为研究新发现的分泌蛋白基因的功能提供了一个非常有用的工具。少

项目成果

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Adachi O、Nakano A、Sato O、Kawamoto S、Tahara H、Toyoda N、Yamato E、Matsumori A、Tabayashi K、Miyazaki J：“通过体内电穿孔进行 Fc 融合细胞因子的基因转移：在病毒性心肌炎基因治疗中的应用

Nakano A, et al.: "Cytokine gene therapy for myocrditis by in vivo electroporation"Hum Gene Ther. 12. 1289-1297 (2001)

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Kobuke K 等人：“ESDN，一种从血管细胞克隆的新型神经毡蛋白样膜蛋白，具有真核生物中最长的分泌信号序列，在血管损伤后表达上调”J.Biol.Chem。