Research of molecular biology and gene therapy for Rheumatoid arthritis by regulation of IL-6 signal transduction

白细胞介素6(IL-6)是类风湿关节炎(RA)患者的炎症活动性指标之一。由大阪大学组织的29个研究所使用人源化的抗IL-6受体抗体(MRA)进行的大规模临床试验证明，用MRA阻断IL-6信号是治疗RA的有效方法。我们还验证了抗IL-6受体治疗的两个关键机制。一种是通过抑制IL-6诱导的血管内皮生长因子(VEGF)的产生来抑制滑膜细胞的血管生成，另一种是通过抑制IL-6诱导的基质金属蛋白酶(MMPs)的过度产生来防止骨和软骨的破坏。我们发现，IL-6与IL-1和肿瘤坏死因子α协同作用，上调血管内皮细胞生长因子和基质金属蛋白酶的产生，从而发挥关键作用。抗IL-6治疗对RA患者血管内皮细胞生长因子和基质金属蛋白酶的抑制效果最好。在n-…中观察到IL-6诱导的血管内皮生长因子产生的增加正常成纤维细胞、滑膜成纤维细胞从OA患者和RA患者中获得较多。我们还发现IL-6可以诱导恶性间皮瘤细胞中血管内皮生长因子的产生。我们通过给IL-6转基因小鼠注射淀粉样增强因子(AEF)建立了继发性淀粉样变性小鼠模型。抗小鼠IL-6受体抗体的治疗减轻了这些小鼠的淀粉样变性，这意味着IL-6是继发性淀粉样变性发生的必要分子。IL-6细胞因子信号的负调控因子SOCS1和SOCS3可用于抗IL-6治疗。我们评估了实现SOC有效表达的最佳基因转移条件。尽管腺病毒对成纤维细胞的感染性较低，但感染性增强型腺病毒在其纤维区具有RGD基序，获得了对滑膜成纤维细胞的高感染性，并克服了这一缺点。我们还用炎症特异性启动子取代了通用启动子(CMV)来控制转基因在RA滑膜成纤维细胞中的表达。我们获得了含有人IgG1Fc的单链重组抗体。该分子成功地抑制了IL-6依赖的细胞生长，并抑制了IL-6刺激诱导的STAT3的磷酸化。这种药物编码在单基因上，很容易应用于病毒基因转移的方法。该分子有望成为抗IL-6治疗与基因治疗相结合的潜在靶点。较少

Interleukine 6 (IL-6) is one of the inflammatory active indexes in rheumatoid arthritis (RA) patients. A massive clinical trial using a humanized anti-IL-6 receptor antibody (MRA), involving 29 institutes under the organization of Osaka University, has provedn that IL-6 signal blocking by MRA is an efficacious treatment for RA. We have also verified two critical mechanisms for the anti-IL-6 receptor therapy. One is anti-angiogenic activity in synoviocytes by means of the inhibition of IL-6-induced vascular endothelial growth factor (VEGF) production, and the other is preventing the destruction of bone and cartilage by inhibiting IL-6-induced matrix metalloproteinase (MMP) over-production. We have found that IL-6, synergistically with IL-1 and TNF α, up-regulates VEGF and MMP production and therefore plays a pivotal role. The anti-IL-6 treatment appeared most efficacious for the suppression of VEGF and MMP in RA patients. Augmentation of VEGF production induced by IL-6 was observed in n … More ormal fibroblasts, synovial fibroblasts obtained from OA patients as well as RA. We also found that IL-6 induced VEGF production in malignant mesothelioma cells.We established secondary amyloidosis mouse models by virtue of the administration of amyloid enhancing factor (AEF) to the IL-6 transgenic mouse. The treatment of an anti-mouse IL-6 receptor antibody mitigated the amyloidosis in these mouse models, which means that IL-6 is an essential molecule for the development of secondary amyloidosis.SOCS1 and SOCS3, negative regulators of IL-6 cytokine signal, are available for the therapeutic agents as anti-IL-6 therapy. We evaluated optimal gene transfer conditions to achieve the effective expression of SOCS. Although the adenovirus has shown low infectivity to fibroblasts, infectivity enhanced adenoviruses having RGD motifs in their fiber region acquired high infectivity to the synovial fibroblasts and overcame this disadvantage. We also replaced the universal promoter (CMV) with inflammation specific promoters for the control of the transgene expression in RA synovial fibroblasts.We generated a single chain recombinant antibody comprising human IgG1 Fc genetically fused to a single chain Fv derived from the parent antibody MRA. This molecule successfully reduced the IL-6-dependent cell growth and inhibited the phosphorylation of STAT3 induced by IL6 stimulation. This therapeutic agent, encoded on single gene, is easily applicable to the viral gene transfer method. This molecule should be a potential device for the anti-IL-6 therapy combined with the gene therapy method. Less