1. ¹William S. Rowe Division of Rheumatology, Children's Hospital Medical Center, Cincinnati, OH 45229, USA
2. ²Division of Rheumatology, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
3. ³Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, OH 45229, USA
4. ⁴Institute of Human Gene Therapy, University of Pennsylvania Health System, M6.30 Maloney Building, Philadelphia, PA 19104-4283, USA

Correspondence: Raphael Hirsch, Division of Rheumatology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: (412) 692 5054. E-mail: raphael.hirsch@chp.edu

^*These authors contributed equally to this work.

Received 14 September 2004; Accepted 24 October 2004.

Abstract

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 1-anti-trypsin under the control of the chick -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.