| Rheumatoid Arthritis(RA) is a kind of chronic autoimmune disease that mainly affects joints and is characterized by infiltration of inflammatory cells and synovial hyperplasia, resulting in the destruction of cartilage and bone. The pathogenesis of RA is complex, which involves many factors including TNF-α, one of the key inflammatory cytokines, as well as auto-reactive T cells. Both of them play crucial role in the process of RA. Biotherapy, especially TNF blockade, has made great achievement in clinical treatment of RA. Fusion protein of p75 tumor necrosis factor receptor and Ig G1 Fc(TNFR-Fc) has been proved as an effective biotherapeutic method. However, some research argued that this treatment is invalid for a substantial number of patients. Therefore, other adjuvant and cooperative treatments are compelling need for RA patients.CD4+ T cells are mainly present in the inflamed articular cavity and synovium, which play important role in the process of RA. As a result, T cells blockade is an effective strategy for RA therapy. Due to the complex pathogenesis of RA, the combined blockade of different causative factors has been an attractive therapeutic strategy of RA. Based on the previous studies, combination of anti-TNF with anti-T cell therapy could enhance the therapeutic function in the animal models of RA. Some research implies that combined blockade of T cells and TNF appears more effective in treating RA than simply blockade TNF in vivo. Therefore, the combination of TNF blockade and T cells blockade is a promising strategy for RA therapy.Gene therapy refers to the recombination of target genes with therapeutic effects and eukaryotic expression vector. The vector is then transfected to human cells and expresses peptides or proteins with therapeutic effects, which contributes to the relief of a disease. Based on a long-term effective treatment ofgene therapy, we should develop combination gene therapy research of RA. By reason of the local affected joints of RA, local gene therapy could be adopted. Recently, Adeno-associatd virus(AAV) is a suitable gene delivery vector and mostly used in animal studies of RA. AAV2 is currently the common serum type that has been applied to clinical treatment, with good safety and low immunogenicity. Therefore, gene therapy of RA will be studied by means of AAV2.In our previous studies, we successfully constructed CTLA4-Fas L with extracellular domains of CTLA4 and Fas L,by means of AAV2,we proved that CTLA4-Fas L fusion protein couldeffectively suppress adjuvant-induced arthritis in rats by intraarticular gene transfer. Among them,CTLA4 blocks costimulatory signal for T-cell activation and Fas L mediates apoptosis of T cells. CTLA4-Fas L is more effective in blocking the process of arthritis than Fas L alone.Based on our previous study, our research aimed to construct a double fusion molecule of TNFR-Fc and CTLA4-Fas L carried by AAV2. We creatively introduced furin and 2A between the two fusion genes, which could regulate the simultaneous, balanced and separate expression of two antiarthritic molecules within a single vector and a single open reading frame in local joints. And we investigate whether the combination of TNFR-Fc with CTLA4-Fas L would produce a more effective suppression of joint inflammation in rat experimental model of RA, and explore the possible mechanisms of this combination therapy.First of all, we inserted TNFR-Fc fusion gene fragments into AAV2 vector, which was named as TRFC, and then tested its secretory expression in cell model in vitro and identified the biological neutralizing activity of rat TNFα. Next, by overlap PCR, we inserted furin cleavage site and 2A self-processing sequence into CTLA4-Fas L fusion gene which has suppressive effect on activated T cells, then furin-2A-CTLA4-Fas L fusion gene with the restriction enzyme site was inserted into TRFC recombinant plasmid, thus double fusion molecules AAV2.TNFR-Fc(furin 2A)CTLA4-Fas L(named TFCF) that we desired was obtained. Then the TFCF recombinant plasmid was transfected into HEK-293 T cells, and we tested the serum-free supernatant of transfection by Western Blot and ELISA to verify independent coexpression of TFCF recombinant plasmid. Finally, recombinant adeno-associated virus(AAV) serotype 2 vectors were produced and were injected in AIA rat ankle joints. The clinical signs of inflammation in paws, ankle or wrist joints were observed by day 11 postinduction to explore the possible mechanisms of this combination therapy by a number of detection of methods such as immunohistochemistry, real-time quantitative PCRand ELISA.The results showed that(1) p AAV2.TNFR-Fc recombinant plasmid was obtained and the secretory expression of fusion gene TNFR-Fc was verified in vitro, which had biological neutralizing activity of rat TNFα to L929 cells.(2) p AAV2.TNFR-Fc(furin 2A) CTLA4-Fas L(named TFCF) was obtained and we had verified the independent coexpression of TNFR-Fc and CTLA4-Fas L in TFCF recombinant plasmid.(3) We observed the expression and therapeutic effects of double fusion molecule in experimental rats in vivo, and the results showed that furin and 2A self-processing polypeptides could regulate the simultaneous and separate expression of the upstream and downstream genes of double fusion molecule.Through the observation of clinical morphology index such as articular index and ankle swelling thickness, and histologic index obtained by HE staining and safranin O-fast green staining, we found that double fusion molecular was more effective in decreasing the swelling degree of ankle and inhibiting inflammatory in arthritis rats than that of the single fusion molecular.Finally,we explored the possible mechanisms of this combination therapy. We detected the inflammatory cytokines, and the results demonstrated that the double fusion molecules could obviously promote the secretion of anti-inflammatory cytokines and suppress that of proinflammatory cytokine, so as to control the inflammatory development.We investigated the extent of infiltrating CD4+ T cells in the joints of arthritic rats through immunohistochemistry detection of intra-articular CD4 gene and TNFα gene levels. Statistical analysis demonstrated that TNFR-Fc/CTLA4-Fas L combination was significantly superior to TNFR-Fc or CTLA4-Fas L alone in prohibiting the infiltration of inflammatory cells including pathogenic CD4+ T cells into the joints, and more effectively down-regulated the expression level of TNFα in joints. Then we isolated the draining inguinal and popliteal lymph nodes, and processed them for analysis of the presence and frequency of CD4+CD25+Fox P3+ Treg cells in these DLNs by flow cytometry, and the data showed that combined therapy induced statistically significant increase in those of Treg cells, which indicated the synergistic antiarthritic effect of the combination of CTLA4-Fas L and TNFR-Fc. These results partially depended on the immunoregulatory mechanism associated with upregulating the levels of CD4+CD25+Fox P3+ Treg cells in the DLNs. We next investigated the regulatory effect of the combination therapy on the splenic inflammatory responses, and the results suggested that the combination treatment can more effectively inhibit the splenomegaly and significantly reduced LPS-stimulated proliferation of spleen cells and nitrite level in spleen cells from AIA rats.In this study, we used the furin-2A fusion sequence to mediate TNFR-Fc and CTLA4-Fas L expression from a single ORF, which enabled us to construct a coexpression system by single r AAV vector for the production of two desirable arthritis inhibitory fusion molecules TNFR-Fc and CTLA4-Fas L. Through a variety of morphological and histological indexes, relevant detections such as ELISA for inflammatory factor, synergistic anti-inflammatory effect of the novel double fusion molecules had been proved, and this expression system presented a feasible gene therapy approach for long-term delivery of TNFα antagonist and T-cell antagonist in vivo in RA. The combination therapy offered a novel potential therapeutic strategy for RA treatment and provided theoretical and experimental basis for clinical research and development of new drugs. |
PDF Full Text Request