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）患者炎症活性指标之一。由大阪大学组织的29个研究所参与的人源化抗IL-6受体抗体（MRA）大规模临床试验证实，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活性，另一种是通过抑制il -6诱导的基质金属蛋白酶（MMP）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TNF α协同上调VEGF和MMP的产生，因此发挥了关键作用。抗il -6治疗对抑制RA患者VEGF和MMP最有效。在OA患者和RA患者的正常成纤维细胞、滑膜成纤维细胞中观察到IL-6诱导的VEGF生成增加。我们还发现IL-6诱导恶性间皮瘤细胞中VEGF的产生。我们通过给IL-6转基因小鼠注射淀粉样蛋白增强因子（AEF）建立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模型中的淀粉样变，这意味着IL-6是继发性淀粉样变发生的必要分子。SOCS1和SOCS3是IL-6细胞因子信号的负调节因子，可作为抗IL-6治疗药物。我们评估了实现SOCS有效表达的最佳基因转移条件。虽然腺病毒对成纤维细胞的感染性较低，但在纤维区具有RGD基序的传染性增强腺病毒对滑膜成纤维细胞具有较高的感染性，克服了这一缺点。我们还用炎症特异性启动子替换通用启动子（CMV）来控制RA滑膜成纤维细胞中的转基因表达。我们产生了一种单链重组抗体，包括人IgG1 Fc基因融合到来自母抗体MRA的单链Fv。该分子成功降低il -6依赖性细胞生长，抑制il -6刺激诱导的STAT3磷酸化。该治疗剂在单基因上编码，易于应用于病毒基因转移方法。该分子可作为抗il -6联合基因治疗的潜在手段。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