A Combinatory Strategy For Gene Therapy Against OA:in Vivo Transfer Of IL-Ra/HSV-TK Gene In Osteoarthritic Rabbit Knee Joints

ObjectiveOsteoarthritis(OA) is one of the high incidence of disease in older population, in the combination with whole body factors and with local factors, articular cartilage biochemical, structure and metabolic happened changes, eventually appear articular cartilage softening, burst and local peeling and bone and cartilage edge joint formation vegetations pathological changes. It can produce clinical joint pain and restricted movement and joint deformities and other symptoms and seriously influence the health of old people, is the important reason for the disabled. In OA, excessive release of proinflammatory cytokines attributes greatly to the pathological changes in local joint. Among these cytokines, IL-1and TNF-a are the most important of cytokines in the pathogenic process, which promote cartilage matrix destruction and articular cartilage degradation. Other cell factors on the destruction of articular cartilage is often indirectly by IL-1and TNF-a. In present so many research about the pathogenesis and treatment of OA, but the effect is an unsatisfying, in recent years suicide gene therapy offers a radical different approach to the treatment of osteoarthritis. This study demonstrates that direct in vivo transfer of the exogenous anti-inflammatory cytokine gene combined with suicide gene by rAAV into osteoarthritic rabbit knee joints using intra-articular injections to observe the therapeutic effect of osteoarthritis.This avenue may therefore represent a promising future treatment for osteoarthritis. MethodsOA was induced in right knees of32New Zealand white rabbits by the excision of medial meniscectomy plus the anterior cruciate ligament transaction. Approximately1×1010v.p./ml of rAAV were injected into the joint space of the knee through the patellar tendon. A total of32operated rabbits were divided into four groups. Four rabbit groups received rAAV encoding IL-1Ra gene as Group A, HSV-tk gene as Group B as well as combined-gene transferred Group C and the control group D, being transfected by the rAAV carrying no target gene. Twenty-four hours following intra-articular injection the animals received intravenous infusions of ganciclovir (GCV) at a close of5mg/kg. The ganciclovir was administered twice daily for a total of3days. One week after the rAAV injection, keen joints were lavaged with1ml of0.9%(w/v) physiological saline solution through the patellar tendon. Concentration of exogenous IL-1Ra was determined by ELISA.Two weeks after the rAAV injection, the rabbits were sacrificed. Keen joints were lavaged with1ml of0.9%(w/v) physiological saline solution through the patellar tendon. Concentration of IL-1Ra、 IL-1β、TNF-a and TGF-β1expression in recovered lavage fluids were measured using a cytokine ELISA kit.. Levels of IL-1Ra,IL-1β,TNF-a, TGF-β1and HSV-tk expression in synovium were measured using rear-time PCR. Gene expressions in all samples were analyzed by applying the2-AACt relative quantification method. Cartilage from the lesion areas of medial femoral condyle were fixed, embedded, sectioned and stained with hematoxylin and eosin and Safranin O. The samples were examined by light microscopy. Histologic quanitative assessment was performed by Mankin’s score.ResultsConcentration of exogenous hIL-1Ra was determined by ELISA assays. At seven days and14days after gene transfection, the exogenous hIL-1Ra was expressing stability in the synovial fluid, while the transfection of the B group of the HSV-TK gene and group D no gene transfection control group does not detect the expression of human IL-1Ra. The exogenous gene can express in the intra-articular.According to the measurement of real-time PCR and the analysis of relative quantification-comparative CT method, expression levels of IL-lβwere significantly decrease in group A,B and C than in group D. The expression levels of TNF-a were significantly increase in group D than in in group A,B and C. The expression of TGF-β1,all of the gene transfection groups showed significant decreases compared with the non-transfected group.Concentration of exogenous hIL-1Ra was determined by ELISA assays. The expression of hIL-1Ra was high following hIL-1Ra gene transfection in synovial fluids. Such trends were also notable at protein level for IL-1β、TNF-a and TGF-(31by ELISA assays. The level of IL-1β was markedly higher in group D than group A,B and C. The level of TNF-a was lower in gene transfection groups than group D. The level of TGF-β1was lower in gene transfection groups than group D. The results of ELISA was shown as mean±SEM, n=3and P<0.05. In the control group, the articular cartilage surface was rough and had some superficial leakage and ulcers. After exogenous genes were transfected, the cartilage lesions and matrix degradation were less evident.Conclusions:The target gene were successfully transferred to the joint space of the knee in vivo carried by rAAV. The transfected genes were expressed in synovial fluid of rabbits. This study suggests that combination-gene transfection show better results than transfection gene alone, gene transfection is effective in treating experimental OA. Combination-gene transfection has better biologic effects on OA.