Proteasome inhibition enhances AAV-mediated transgene expression in human synoviocytes in vitro and in vivo.

To explore the potential applicability of recombinant adeno-associated virus (rAAV) vectors in the treatment of rheumatoid arthritis (RA), primary human fibroblast-like synoviocytes (FLS) derived from patients with RA were infected with rAAV encoding mouse IL-10 under the control of the CMV promoter. Addition of the proteasome inhibitor carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (zLLL) to the cultures dramatically enhanced expression of the IL-10 transgene, in a dose-dependent manner. The increased expression was transient, peaking at 3 days and returning to near baseline by 7 days. The enhancement was observed even when zLLL was added 13 days after infection with rAAV. The effect of zLLL was not specific to either the mIL-10 transgene or the CMV promoter, as similar findings were observed using an rAAV construct encoding alpha1-anti-trypsin under the control of the chick beta-actin promoter or GFP, driven by the CMV promoter. Transgene expression could be repeatedly induced by reexposure to zLLL. Transgene mRNA levels increased in parallel with protein levels. Transgene expression could also be repeatedly induced in vivo by administering zLLL to SCID mice previously injected with rAAV-infected FLS. These data demonstrate that proteasome inhibition can dramatically enhance transgene expression in human RA FLS following infection with rAAV and suggest a possible approach to regulating synovial transgene expression in vivo.

Recombinant adeno-associated virus preferentially transduces human, compared to mouse, synovium: implications for arthritis therapy.

Despite a number of published reports, including from our own laboratory, suggesting that adeno-associated virus (AAV) transduces mouse synovium, a careful analysis demonstrated transduction predominantly of the subsynovial muscle tissue, while the synovial lining is poorly transduced. To investigate the potential of AAV to transduce human synovium, three human rheumatoid arthritis (RA) and two murine collagen-induced arthritis (CIA) synovial cell lines were infected with recombinant AAV (rAAV) vectors encoding either mouse IL-10 or IL-4. Low-level transgene expression was observed. However, either Gamma-irradiation or the addition of a low-titer E1-, E3-deleted recombinant adenovirus resulted in up to a 100-fold increase in transgene product in the human, but not the mouse, cell lines. RA synovial tissues implanted subcutaneously in severe combined immunodeficiency (SCID) mice, which were subsequently infected with rAAV, showed marked increases in transgene expression when co-infected with adenovirus. To our knowledge, this is the first study to show that intact human synovial tissues can be transduced by rAAV, and it suggests that murine arthritis may not be an optimal model to study rAAV as a gene transfer vector. Further studies to elucidate the mechanisms limiting gene transduction in human synovium may allow optimization of this vector for the treatment of arthritis.