93. Macrophages Are a Barrier to AAV5 Mediated Gene Expression in the Air Pouch Synovial Inflammation (APSI) Model

Abstract

Our group is investigating the application of AAV gene therapy for inflammatory disease, especially rheumatoid arthritis (RA). The air pouch synovial inflammation (APSI) model is commonly used for studying local inflammation in rodents and is often used to test anti-rheumatic compounds. It involves the subcutaneous injection of air in the back of the animal, promoting the formation of an air pouch lining. After ~6 days, this lining bears a strong resemblance to the synovial membrane of a joint, being composed primarily of fibroblast-like and macrophage-like cells. Inflammation can be induced by injecting a stimulus (e.g. LPS, TNF, synovial fluid) into the air pouch cavity. While not a true RA model, the APSI model has a number of advantages over standard mouse arthritis models, including a short time frame (2 weeks versus 2 months), low discomfort for the animals, and allows for analysis of inflammatory markers in the air pouch fluid and membrane. Additionally, many of the treatments that have proved effective in human RA (e.g. methotrexate, TNF-blockers, steroids) are also effective in the APSI model.In order to use the APSI model for testing new gene therapies for RA, we investigated the kinetics of AAV5 mediated gene expression. An AAV5-CMV-Luciferase vector was used to determine optimal vector doses by non-invasive in vivo whole animal bioluminescent imaging. To our surprise, when vector was injected at day 6 after formation of the air pouch, we were unable to detect any luciferase expression up to day 30, over a wide range of vector doses (1×10E9 to 1×10E12 vg). Subsequent studies confirmed that similarly to synovial fibroblasts, air pouch fibroblasts were able to be transduced by AAV5 in vitro.As immunohistochemistry (IHC) staining (F4/80) revealed a high density of macrophages in the air pouch lining at day 6, we hypothesized that the presence of these cells might be inhibiting AAV5 mediated transduction in the APSI model. To test this hypothesis we either suppressed macrophage activity using triamcinolone (steroid) in combination with AAV5-CMV-Luciferase injection at day 6, or avoided macrophages by administering vector before the influx of macrophages into the air pouch lining (day 0). We observed that either suppressing or avoiding macrophages allowed for expression of luciferase following AAV5-CMV-Luciferase administration.These data suggest that macrophages are a previously underappreciated barrier to gene transfer, and that strategies, such as immune suppression or macrophage depletion, should be explored further when administering vector to macrophage rich tissue.