The long-term objective of this project is to determine if genetically engineered therapeutic agents delivered to rheumatoid synovial cells and/or rheumatoid synovium are capable of controlling cartilage degradation in rheumatoid arthritis (RA). At present the therapeutic agents used to treat RA have not been shown to effectively block the disease process. Identification of novel therapies which ameliorate the disease would be of valuable clinical utility. An emerging potentially fruitful therapeutic area for treatment of RA entails the introduction of gene therapy agents into RA synovium that are specifically designed to block the disease process. Clinically speaking, glucocorticoids have been beneficial in treatment of RA but their use is limited because of their undesirable systemic side effects. There is strong evidence that glucocorticoids repress the transcription of several key genes that ultimately cause the degradation of cartilage in RA. These genes include: collagenase, stromelysin, IL-1beta and TNF-alpha. Introducing a mutant glucocorticoid receptor into RA synovium which has the beneficial effector of glucocorticoid therapy without the undesirable systemic side effects would be a major therapeutic advantage. To achieve the objectives of this project, three specific aims are proposed: 1) Determine conditions for transfection of model synovial cells with genetically engineered mutant glucocorticoid receptors and measurement of mutant receptor expression. Mutant receptors will be delivered to cells with lipofectin or adeno-associated virus and their expression measured by RT-PCR, filter binding and sucrose gradients. 2) Determine if genetically engineered mutant glucocorticoid receptors complexed with ligand RU486 can suppress the transcription of collagenase and stromelysin genes in model synovial cells. Repression of protease gene transcription will be measured by Northern blots and quantitative RT-PCR. 3) Determine the role that mutant glucocorticoid receptors play in controlling the macromolecular turnover of cartilage-derived collagen and proteoglycans. Degradation of type II collagen will be measured by hydroxproline release while degradation of proteoglycans will be measured by release of 35Sulfate from prelabeled 35S-proteoglycans. Hopefully, these studies will lead to improved treatment of RA through application of novel gene therapy approaches.
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