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）是类风湿性关节炎（RA）患者的炎症活性指标之一。一项使用人源化抗IL-6受体抗体（MRA）、涉及大阪大学下属29个研究所的大规模临床试验证明，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) 产生来发挥滑膜细胞的抗血管生成活性，另一种是通过抑制 IL-6 诱导的基质金属蛋白酶 (MMP) 过量产生来防止骨和软骨的破坏。我们发现 IL-6 与 IL-1 和 TNF α 协同作用，上调 VEGF 和 MMP 的产生，因此发挥着关键作用。抗 IL-6 治疗似乎对抑制 RA 患者的 VEGF 和 MMP 最有效。在从 OA 患者以及 RA 获得的正常成纤维细胞、滑膜成纤维细胞中观察到 IL-6 诱导的 VEGF 产生增加。我们还发现IL-6诱导恶性间皮瘤细胞产生VEGF。我们通过给IL-6转基因小鼠注射淀粉样蛋白增强因子(AEF)来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性，这意味着IL-6是继发性淀粉样变性发展的重要分子。SOCS1和SOCS3是IL-6细胞因子信号的负调节因子，可用作抗IL-6疗法的治疗剂。我们评估了实现 SOCS 有效表达的最佳基因转移条件。尽管腺病毒对成纤维细胞显示出低感染性，但在其纤维区域中具有RGD基序的感染性增强的腺病毒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），以控制 RA 滑膜成纤维细胞中的转基因表达。我们生成了单链重组抗体，其中包含与源自亲本抗体 MRA 的单链 Fv 基因融合的人 IgG1 Fc。该分子成功减少了 IL-6 依赖性细胞生长，并抑制了 IL6 刺激诱导的 STAT3 磷酸化。这种由单基因编码的治疗剂很容易适用于病毒基因转移方法。该分子应该是与基因治疗方法相结合的抗IL-6治疗的潜在装置。较少的

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