Proteasome Inhibition Enhances AAV-Mediated Transgene Expression in Human Synoviocytes in Vitro and in Vivo

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 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.

The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

“…This effect has been attributed to activation of the cellular DNA damage response, which in turn is important to process the vector genome to generate a transcriptioncompetent template (Cervelli et al, 2008). The development of capsid variants optimized for cell targeting and/or trafficking (Bü ning et al, 2008;Li et al, 2008), as well as the use of compounds that do not directly damage the DNA, such as proteasome inhibitors ( Jennings et al, 2005;Monahan et al, 2010), have further improved AAV transduction efficiencies both in cultured cells (Ellis et al, 2012) and in vivo (Paulk et al, 2012). The effect might be linked to the nuclear trafficking of the viral particles.…”

Down‐regulation of cathepsin K in synovium leads to progression of osteoarthritis in rabbits

“…As mentioned above, in this study we focused on the functional analysis of cathepsin K as a candidate gene associated with OA pathogenesis. Gene expression in synovium can be easily manipulated, as compared with that in cartilage (19,20). Recently, it was recognized that use of siRNA makes it possible to perform specific gene-silencing experiments in vivo (21)(22)(23).…”

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

“…MG 132 is a low-molecular-weight molecule that is among the most widely used peptide aldehyde inhibitors of the proteasome. It readily enters cells in vitro and retards proteasomal destruction of ubiquitinated proteins by inhibiting proteolysis (25)(26)(27)(28). Consequently, MG132 has been employed to identify conditions in which altered expression of cellular proteins occurs through changes in proteasomal function.…”