Protection Of Human Osteoprotegerin Mediated By Adeno-associated Virus On Bone Erosion In Collegen-induced Arthritis

Background:Rheumatoid arthritis(RA) is a systemic autoimmunity disease,characterized by progressive synovial inflammation and joint destruction.Most reasearches indecate bone destruction involves osteoclasts,the specialized cells that resorb calcified bone matrix. RANK ligand(RANKL) expressed by stromal cells and T cells,and its cognate receptor, RANK,were identified as a critical ligand-receptor pair for osteoclast differentiation and survival.A decoy receptor for RANKL,osteoprotegerin,(OPG) impinges on this system and regulates osteoclast numbers and activity.RANKL is also expressed in collagen-induced arthritis(CIA) in which focal collections of osteoclasts are prominent at sites of bone destruction.Viral mediated gene transfer is currently the most efficient system for delivering therapeutic proteins in vivo.Adeno-associated virus(AAV) especially holds great promise as a new vector and appears to be safe.AAV is a single stranded DNA virus that does not induce a significant immune response and is not associated with disease in humans.The ability of AAV to mediate expression of therapeutic genes has been well established in several animal models of arthritis.Recombinant AAV2(rAAV2) has been validated for gene therapy in human clinical trials in cystic fibrosis,a 1antitrypsin deficiency,and haemophilia,rAAV vectors are particularly useful in targeting slowly dividing cells and in the treatment of chronic disease because of their potential for site-specific integration into the host genome or formation of stable episomal DNA,both of which result in long term gene expression.In this study,we have established AAV mediated hOPG gene transfer system and demonstrated that rAAV-OPG can infect the tissue of joint.we investigated protection effects on joint destruction by rAAV-hOPG in CIA.Partâ… Estanblishment of rAAV2-hOPG and Titre DeterminationObjective:Establishment of the rAAV-hOPG,M(?)thods:Establish recombination pSNAV2.0-hOPG plasmid,then use it transfects BHk-21 cell,adenovirus,rAAV-hOPG were prepared by series of procedures of picking plaques,amplification,purification and identification.Results:Prepared rAAV-hOPG with high titre(4×10¹¹vg/ml).Conlusions: The preperation of rAAV-hOPG(with high titre) is reliable for the further research work.Partâ…¡Protection of Bone Erosion in Collegen-induced Arthritis by rAAV-hOPGObjective:Using an in vivo adeno-associated virus(AAV)-mediated gene transfer technique,this study was designed to evalute the protect effects of OPG transgene against joint destruction in CIA model.Methods:After induction of CIA in Sprague-Dawley rats, test animals were treated with PBS or rAAV-EGFP or rAAV-hOPG(100ul/day^*10d) intra-articular injection for 10 days,beginning at the onset of disease.Paraffin-embedded joints were then analyzed histologically.Osteoclasts were identified using TRAP staining.The joint destrution was evalued by Lasen Score.The synovium expression of mRNA for OPG,TRAP and VEGF was identified by real-time polymerase chain reaction (PCR).The protein expression of OPG,IL-1,MMP-3 was identified by enzyme-linked immunosorbent assay(ELISA).Results:Successful transgene expression was confirmed by the dectection of OPG by ELISA and positive fluorescence of frozen section,Real-time PCR indicated significant diminishment of messenger RNA expression of osteoclast markers in OPG transduced group compared with rAAV-EGFP transduced group and CIA group.The transduction and expression of OPG hasn't markedly affected the gene copies of VEGF.Image analysis revealed that expression of OPG significantly protected against joint destruction compared with CIA group.by 30.18%.Conclusion:OPG gene transfer mediated by rAAV effectively protects against bone destruction induced by CIA model. Data suggest that gene transfer using rAAV-OPG may be a feasible and effective therapeutic candidate to treat or prevent joint destruction in inflammatory arthritis.