Cytokine gene therapy of cardiovascular diseases by in vivo electroporation

在众多体内基因转移技术中，将质粒DNA直接注射到肌肉中简单、便宜且安全。我们尝试通过转移抑制性细胞因子IL-1受体拮抗剂（IL-1ra）或病毒KL-10（vIL-10）来治疗病毒性心肌炎，通过体内电穿孔，这是一种将基因转移到肌肉的新方法，对4周龄的雄性DBA/2小鼠腹膜内接种10 PFU脑心肌炎病毒。病毒接种后，立即将携带IL-1ra或vIL-10的表达质粒注射到胫骨前肌中，然后进行电穿孔。第5天，IL-1ra和vIL-10的血清水平分别达到10.5和2.3 ng/ml，此时基因表达达到峰值。组织病理学检查显示，与对照组相比，IL-1ra或vIL-10处理的小鼠心肌细胞浸润得到改善。心肌炎发病后第14天，转入IL-1ra或vIL-10表达质粒显着提高了动物的存活率。 IL-1ran处理后，TNF-α的表达降低至0.60倍（p < 0.005），诱导型一氧化氮合酶（iNOS）表达降低0.43倍（p < 0.005），心脏中IFN-γ的表达降低至0.35倍（p < 0.05），iNOS表达降低0.21倍（p < 0.05）。 0.005），相对于对照，vIL-10。为了提高基因转移的效率，我们开发了一种质粒载体，将分泌蛋白表达为与非细胞溶解性免疫球蛋白Fc部分的融合蛋白，并将其用于电穿孔介导的vIL-10体内表达。融合细胞因子vIL-10/mutFC被成功表达，并且vIL-10的血清峰浓度比非融合vIL-10表达质粒几乎高100倍(195ng/ml)。表达的融合细胞因子降低了小鼠病毒性心肌炎模型的死亡率。这些结果表明，表达非溶细胞性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。这种简单而有效的系统应该为人类疾病的基因治疗提供一种新方法，并且对于研究新发现的分泌蛋白基因的功能非常有用。较少的

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