Highly Efficient Gene Expression in Rabbit Synoviocytes Using EBV-Based Plasmid

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic synovial inflammation which leads to joint destruction. Gene therapy of RA targets the players of inflammation or articular destruction. However, viral vectors have safety problems and side effects, while non-viral vectors suffer from inefficient gene transfer and fast loss of gene expression. To overcome the limits of non-vial vectors, an EBV-based plasmid which is known to exert prolonged high level gene expression can be used. METHODS: pEBVGFP, pEBVIL-10, and pEBVvIL-10 were constructed by cloning GFP, IL-10, and vIL-10 genes into an EBV-based plasmid, respectively. The pGFP was used as a control plasmid. Each constructs were lipofected into HIG-82 rabbit synoviocytes. The expression of GFP was monitored by FACS and confocal microscopy. IL-10 and vIL-10 expressions were measured by ELISA. RESULTS: GFP expression 2 days after transfection was achieved in 33.2% of cells. GFP-expressing cells transfected with pGFP decreased rapidly from 4 days after transfection and disappeared completely by 11 days. Cells transfected with pEBVGFP began to decrease slowly from 4 days. But GFP expression was detected for over 35 days. In addition, HIG-82 cells transfected with pEBVIL-10 (44.6+/-1.5 ng/ml) or pEBVvIL-10 (51.0+/-5.7 ng/ml) secreted these cytokines at high levels. High level cytokine production by hygromycin selection was maintained at least for up to 26 days after transfection. CONCLUSION: These results suggest that the EBV-based plasmid has a potential to improve non-viral gene transfer system and may be applicable to treat RA without the drawbacks of viral vectors.

Study on the animal model of vIL-10 transgenic therapy for rabbit arthritis / 中国免疫学杂志

Objective:To establish a local ex-vivo gene transfer method to treat RA through animal experiments in vivo and in vitro using retrovirus(rV) as a vector which carrying rRV-vIL-10 target gene.Methods:①The rabbit RA models were induced by the rabbit synovial fibroblast cell line which could continuously expreesed hIL-1?. ②In vivo, the rabbit synovial fibroblast cell line was transduced with rRV-vIL-10, then adding G418 to pick out the rRV-vIL-10 positive clon and infecting the rabbit synovium through intra-articular injection. ③RT-PCR, IHC methods were performed to prove the success of gene transfer to the rabbit synovium and expressed the target protein. ④The relative cytokins changes were detected by ELISA before and after gene therapy and evaluated treatment efficacy of rRV-vIL-10.Results:①rRV-vIL-10 was a effective vector which could transfect to the rabbit synovium in vivo through RT-PCR and IHC methods. ②Intra-articular local gene therapy could effectly reduce the synovium inflammation level of rabbit joints and expressed mRNA and vIL-10 protein. The level of cytokin such as IL-1? was decline.Conclusion:Retrovirus-mediated transgene of vIL-10 is successfully transfected to the rabbit synovium ex-vivo and can reduce arthritis inflammation levels of the IL-1? induced arthritis.