Objective: To evaluate the utility of the adeno associated viral (AAV) vector for gene delivery to joint cells in vivo and in vitro, and to assess its potential as a vector for arthritis gene therapy.
Methods: A recombinant AAV (rAAV) vector expressing the bacterial beta-galactosidase (beta-gal) gene (rAAV-CMV-LacZ) was directly introduced into healthy-normal mouse knees, or arthritic knees in mice overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg). Beta-gal expression levels were determined by immunohistochemistry and chemiluminescence. The transduction efficiency of this vector on primary fibroblast-like synoviocytes (FLS) in vitro was determined by FACS. The effects of UV and gamma-irradiation as well as TNF-alpha on transduction efficiency were determined using the same methods.
Results: We found little evidence of rAAV transduction in the joint cells of healthy mice. Target gene expression was detected in all animals at Day 3, and peaked at Day 7 before returning to baseline levels 21 days after injection. In contrast, synoviocytes, articular chondrocytes, and meniscal cells of diseased mice were transduced by rAAV-CMV-LacZ in hTNFalpha-Tg animals. Transduction efficiencies correlated with joint damage, and target gene expression was up to 10-fold greater than that seen in the normal mice. In vitro, we found that rAAV transduction of FLS can be enhanced by pretreatment with UV or gamma-irradiation and TNF-alpha stimulation.
Conclusion: We find that rAAV vectors have several empirical advantages for in vivo gene therapy for arthritis: (1) rAAV preferentially transduces arthritic joint cells in vivo. (2) rAAV can transduce both FLS and chondrocytes in vivo. (3) rAAV transduction of FLS can be augmented by pretreatment with agents that induce DNA repair enzymes.