| Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory disease of unknown etiology. Cytokines network plays an important role in RA pathological process is generally accepted. Tumor necrosis factor α (TNF α ) is a multifucntion cytokine with activities ranging from host defense mechanisms in infections and injury to severe toxicity in autoimmune disease including RA. Clinical symptomatic relief of RA could be achieved through TNF α receptor binding blockage by biotechnology in recent years. But these proteins are systematic administration, high cost and short dosing interval drugs, and these drawbacks limit their use. However, therapeutic protein level raised by local administration, long term gene expression in vivo, and relative long dosing interval are all predominance of gene therapy. Gene therapy has become one of most promising therapeutic tools of RA due to above predominance.Adeno-associated virus (AAV) is one of most ideal vectors for gene therapy due to its low immunogenicity, ability to infect various types of cells from different tissue origin, and the capability to mediate long term gene expression. Expression level and tansduce efficiency of AAV serpotype vectors are different in various tissue due to capsid disparity.In our study, firstly AAV1, AAV2 containing human or rat tumor necrosis factor receptor- IgGlFc fragment fusion gene (TNFR:Fc) were constructed.The recombinant virus DNA sequence and related physico-chemical characteristics were conformed to the plan. We confirmed that both AAV1 and AAV2 could mediate fusion gene expression in vitro, but there was no significant difference between AAV serotype 1 and 2 vector in vitro. hTNFR:Fc protein expressed by AAV1,AAV2 could neutralized toxicity of TNF a origin from human, rat and mouse effectively. The toxicity of rat TNF a to L929 cells also could be neutralized by BHK-21 cells supernatants infected by AAVl/ratTNFR:Fc, AAV2/ratTNFR:Fc.AAV1, AAV2 could mediated GFP and ratTNFR:Fc fusion gene expression in rat muscles and synovium in vivo.Muscle expression of AAVl/ratTNFR:Fc, AAV2/rat TNFR:Fc were over 13 weeks, and synovium expression were over 30 days. The toxicity of rat TNF a to L929 cells also could be neutralized by the serum of rats received AAVl/ratTNFR:Fc, AAV2/rat TNFR:Fc muscle injections. Transgene muscle expression mediated by AAV1 are stronger and better than AAV2.But synovium expression mediated by these two vectors have no significant difference. Muscle expression of AAVl/hTNFR:Fc also stronger than AAV2/hTNFR:Fc,but both expression level decreased significantly after 1 week. hTNFR:Fc antibody also could be detected in rats serum.This results suggested that immunoresponse could be induced by human fusion gene expression in rat.Based on above researches, we will investigate therapeutic effects of rAAVl/ratTNFR:Fc, rAAV2/ratTNFR: Fc in arthritis model.Species contribution to rat arthritis and induce approaches including complete freund's adjuvant or collagen were compared next. Collagen induced Lewis rat arthritis model was finally confirmed due to its reproducibility and incidence rate. Animal received bovine collagen II base tail injection at day 0,followed by im virus injection, single or both ia virus injection. We found that AAV2/ratTNFR:Fc single ankle ia group, AAVl/ratTNFR:Fc, AAV2/rat TNFR:Fc both ankle ia group, and AAVl/ratTNFR:Fc, AAV2/ratTNFR:Fc im group all revealed less inflammation or joint destruction compared to control group. Inflammation in virus received group...
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