Haim Burstein

983. Repeat Intra-Articular Administration of AAV2 Vectors to Rat Joints

Top of pageAbstract Rheumatoid arthritis is a chronic autoimmune disease characterized by synovial inflammation, leading to the destruction of cartilage and bone. Previously, we demonstrated that a single intra-articular administration of AAV2-TNFR:Fc vector to rats with experimental arthritis resulted in suppression of disease. For treatment of chronic inflammatory arthritis using intra-articular delivery of AAV2-TNFR:Fc vector, intra-articular re-administrations may be required. In this study, we evaluated if serum anti-AAV2 capsid neutralizing antibodies, elicited after intra-articular administration of AAV2-TNFR:Fc vector to rat joints, affect joint transduction following intra-articular re-administration of an AAV2-FlagLuc vector. AAV2-FlagLuc was administered approximately one, three, nine and twelve months after injection of either vehicle or AAV2-TNFR:Fc vector. Two weeks post intra-articular injection of the AAV2-FlagLuc vector, we measured and compared luciferase enzyme activity in joint tissues of vehicle-treated animals with animals treated first with AAV2-ratTNFR:Fc vector. Intra-articular administration of AAV2-TNFR:Fc vector to rat joints elicited serum anti-AAV2 capsid neutralizing antibody response. Following re-administration with AAV2-FlagLuc, the anti- AAV2 capsid neutralizing antibody levels in the serum were up to 10-fold higher compared with those detected prior to vector re-administration. Luciferase enzyme activity, measured following re- administration of AAV2-FlagLuc vector to vehicle-treated animals, was 25-fold higher compared with the assay background in normal joints, and 12-fold higher in inflamed joints. In contrast, luciferase enzyme activity was not detected in normal or arthritic rat joints that were injected first with AAV2-ratTNFR:Fc and then re-administered with AAV2-FlagLuc. However, using RT-PCR analyses, we observed sustained expression of TNFR:Fc for at least 378 days. These results demonstrate that in rats the levels of serum anti-AAV2 capsid neutralizing antibodies, elicited one to twelve months after AAV2 vector administration to the rat joint, are sufficiently high to inhibit transduction following intra-articular re-administration with a vector of the same serotype. The evidence that TNFR:Fc is expressed in the joints for more than a year suggests that frequent repeat administrations to the joints may not be needed.

880. Suppression of inflammation in a rat model of arthritis following intramuscular administration of AAV-TNFR:Fc vector pseudotyped with AAV type 1 capsid

TNF-alpha antagonists such as anti-TNF-alpha monoclonal antibodies or soluble tumor necrosis factor receptors have proven to be useful therapeutics for the treatment of autoimmune inflammatory diseases including rheumatoid and psoriatic arthritis. Recently, we have demonstrated that local (intra-articular) or systemic (intramuscular) administration of an AAV2-ratTNFR:Fc vector, encoding the rat tumor necrosis factor-a receptor: immunoglobulin (IgG1) Fc fusion gene, to rats with experimental arthritis led to suppression of disease as reflected in decreased inflammatory cell infiltration, pannus formation, cartilage and bone destruction, and mRNA expression of joint proinflammatory cytokines. In the current study we employed a monoarticular arthritis model in rats that allow us to evaluate the long-term effect of TNFR:Fc expression on recurrence of joint inflammation. Monoarticular arthritis was initiated by injection of a small dose of peptidoglycan-polysaccharide polymers (PG-APS) isolated from cell walls of group A streptocci into the hind ankle joints. This resulted in a flare of inflammation that resolved within 5 to 7 days. Thirty-two days later, reactivation of joint inflammation was induced only in the joints previously injured by intravenous injection of subarthropathic dose of PG-APS. Twenty-six days later, rats were administered intramuscularly with either vehicle or with AAV-TNFR:Fc vector pseudotyped with AAV type 1 capsid at a dose of 1E12 DNase-resistant particles (DRP). Four weeks post-vector administration, the mean levels of circulating TNFR:Fc protein was 21.5 μg/mL. PG-APS was then administered intravenously to induce a recurring flare of joint inflammation. As expected, animals that were treated with vehicle developed a significant flare of inflammation in their ankle joints. In contrast, the inflammatory response, measured by joint volume, was completely suppressed in AAV2/1-TNFR:Fc-treated animals. To assess the long-term effect of TNFR:Fc expression on suppression of arthritis in this model, animals are currently being evaluated for another round of remission and reactivation of joint inflammation.