IMMUNOSUPPRESSIVE GENE THERAPY FOR RA

越来越多的证据表明关节在病理生理学中起着积极的作用。关节不是身体其他部位发生的重要系统紊乱的被动受害者，它们可能会在比目前认识到的更大程度上推动疾病。滑膜在类风湿关节炎中起着关节内淋巴结的作用，在这方面可能是至关重要的。这种范式转变的一个结果是更加重视关节本身的治疗，而不是关节外部位的治疗。滑膜中T细胞-间充质细胞的相互作用对于T细胞的激活和炎症的诱导都是特别重要的，但相互协同激活的机制在很大程度上是未知的。这项研究的重点是滑膜T细胞与RA滑膜炎症的关系。(1)共刺激CD28/B7和CD40/CD40L相互作用是T细胞活化和存活所必需的。关节内表达可溶性抑制物CTLA4Ig和CD40Ig可提供局部免疫抑制，从而在不影响宿主免疫的情况下缓解疾病。这一假设将在使用这些抑制剂的基因转移的类风湿性关节炎动物模型中得到验证。(2)我们的研究证实了转录因子核因子-kappaB在滑膜炎症和增生中的关键作用，但导致核因子-kappaB持续激活的机制尚不清楚。T细胞与间充质细胞的直接接触，特别是CD40/CD40L和CD28/B7的相互作用可能是通过CD40Ig和CTLA4Ig的基因传递在滑膜中激活和炎症的关键。(3)动物实验的一个预期结果是，通过CD40Ig和CTLA4Ig的基因传递，在单个关节抑制NF-kappaB的激活和滑膜中的炎症。(3)我们的动物研究的一个意外发现是，抑制单个关节中的核因子-kappaB不仅在治疗的关节中抑制炎症，而且在对侧未治疗的关节中也抑制炎症。这可能有许多治疗分支，但这种效果的基础尚不清楚。在滑膜APC、内皮细胞和巨噬细胞中诱导核因子-kappaB调控的细胞因子和细胞黏附分子可能对滑膜APC、内皮和巨噬细胞的发育至关重要，而滑膜APC、内皮和巨噬细胞中的细胞黏附分子和滑膜APC、内皮和巨噬细胞中的细胞黏附分子可能对滑膜T细胞的促炎性Th1反应的发生至关重要。我们推测，局部抑制核因子-kappaB的激活可能使滑膜和循环T细胞中的Th1/Th2平衡向抗炎Th2亚群转移，从而减轻局部和全身T细胞驱动的炎症。这一假设将在拟议的项目中得到检验。(4)这项提议的目的是测试一种用于设计非免疫原性腺病毒(Ad)载体的新概念。腺病毒载体是唯一适合将基因输送到滑膜的载体，但其免疫原性阻碍了临床应用。由于T细胞的活化对Ad转基因免疫应答的发展起着至关重要的作用，我们将研究通过表达CTLA4Ig和CD40Ig基因来抑制Ad载体免疫原性的可行性。在感染细胞附近创造免疫保护环境应该允许长期表达和重复给药。

There is growing evidence for the active role of the joints in pathophysiology. Instead of the joints being the passive victims of important systemic disturbances occurring elsewhere in the body, they may drive the disease to a much greater extent than is presently appreciated. Synovium, which in RA operates as an intraarticular lymph node, could be critical in this regard. One consequence of this paradigm shift would be to put much greater emphasis on treating the joints themselves, rather than extraarticular loci. T-cell-mesenchymal cell interactions in the synovium are particularly important for both T cell activation and induction of inflammation, but the mechanisms underlying mutual co-activation are largely unknown. The proposed study focuses on the relationship between synovial T cell and inflammation in the RA synovium. (1) Co-stimulatory CD28/B7 and CD40/CD40L interactions are required for T cell activation and viability. Intraarticular expression of soluble inhibitors CTLA4Ig and CD40Ig may provide local immunosupression, thereby alleviating the disease without compromising the host immunity. This hypothesis will be tested in animal models of RA using gene transfer of these inhibitors. (2) Our studies established the pivotal role for the transcription factor NF-kappaB in synovial inflammation and hyperplasia, but the mechanisms causing persistent NF- kappaB activation are not known. Direct T cell-mesenchymal cell contacts, particularly CD40/CD40L and CD28/B7 interactions may be critical for NF-kappaB activation and inflammation in the synovium by gene delivery of CD40Ig and CTLA4Ig. (3) One expected finding of our animal studies was that suppression of NF-kappaB in a single joint inhibiting NF-kappaB activation and inflammation in the synovium by gene delivery of CD40Ig and CTLA4Ig. (3) One unexpected finding of our animal studies was that suppression of NF-kappaB in a single joint inhibited inflammation not only in treated, but also in the contralateral, untreated joints. This may have numerous therapeutic ramifications, but the basis for this effect is not known. The induction of NF-kappaB- controlled cytokines and cell adherence molecules in synovial APC, endothelium, and macrophages may be critically important for development and cell adherence molecules in synovial APC, endothelium, and cell adherence molecules in synovial APC, endothelium, and macrophages may be critically important for development of pro-inflammatory Th1 responses in synovial T cells. We hypothesize that local suppression of NF-kappaB activation may shift the Th1/Th2 balance in synovial and circulating T cells toward the anti-inflammatory Th2 subset, thereby alleviating the local and systemic T cell-driven inflammation. This hypothesis will be tested in the proposed project. (4) Closely related, but distinct objective of this proposal is to test a novel concept for designing non-immunogenic adenoviral (Ad) vectors. Ad vectors are uniquely suitable for gene delivery to the synovium, but its immunogenicity precludes clinical applications. As T cell activation is cental to the development of immune responses to Ad transgene, we will examine the feasibility of suppressing the immunogenicity of Ad vectors by expressing CTLA4Ig and CD40Ig genes. Creating immunoprivileged environment in the vicinity of infected cells should permit long-term expression and repetitive administrations.