IMMUNOSUPPRESSIVE GENE THERAPY FOR RA

越来越多的证据表明关节在病理生理中起着积极的作用。关节不是身体其他部位发生的重要全身性紊乱的被动受害者，它们可能会使疾病发展到比目前认识到的更大的程度。滑膜，在类风湿性关节炎中作为关节内淋巴结运作，在这方面可能是关键的。这种模式转变的一个后果是更重视关节本身的治疗，而不是关节外的部位。滑膜中T细胞与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，但相互共激活的机制在很大程度上是未知的。本研究旨在探讨滑膜T细胞与RA滑膜炎症的关系。(1) T细胞的活化和生存需要共刺激CD28/B7和CD40/CD40L的相互作用。可溶性抑制剂CTLA4Ig和CD40Ig的关节内表达可能提供局部免疫抑制，从而在不损害宿主免疫的情况下减轻疾病。这一假设将通过这些抑制剂的基因转移在类风湿性关节炎的动物模型中得到验证。(2)我们的研究确定了转录因子NF-kappaB在滑膜炎症和增生中的关键作用，但导致NF-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的基因传递激活NF-kappaB和滑膜炎症的关键。(3)我们的动物研究的一个预期发现是，在单个关节中抑制NF-kappaB通过CD40Ig和CTLA4Ig的基因传递抑制NF-kappaB的激活和滑膜炎症。(3)我们的动物研究中一个意想不到的发现是，在单个关节中抑制NF-kappaB不仅在治疗关节中抑制炎症，而且在对侧未治疗关节中也抑制炎症。这可能有许多治疗后果，但这种影响的基础尚不清楚。在滑膜APC、内皮细胞和巨噬细胞中诱导NF-kappaB控制的细胞因子和细胞粘附分子可能对滑膜APC、内皮细胞的发育和细胞粘附分子至关重要，而滑膜APC、内皮细胞和巨噬细胞的细胞粘附分子可能对滑膜T细胞中促炎Th1反应的发展至关重要。我们假设局部抑制NF-kappaB激活可能会将滑膜和循环T细胞中的Th1/Th2平衡转向抗炎Th2亚群，从而减轻局部和全身T细胞驱动的炎症。这个假设将在提议的项目中进行检验。(4)密切相关但不同的是，本提案的目的是测试设计非免疫原性腺病毒（Ad）载体的新概念。Ad载体是唯一适合于基因传递到滑膜，但其免疫原性妨碍了临床应用。由于T细胞活化是Ad转基因免疫反应发展的核心，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特权环境应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.